Method for immunizing an avian species

ABSTRACT

The present invention relates to a method for generating a plurality of different IgY antibodies, by immunizing each of a plurality of avian organisms of the same or different species with a composition comprising a plurality of different antigens, thereby generating a plurality of different IgY antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.12/299,247, now U.S. Pat. No. 8,859,233, entitled “Method for Immunizingan Avian Species” and filed on Jun. 4, 2009, which is a national stagefiling under 35 U.S.C. § 371 of international applicationPCT/DK2007/000211, filed on May 1, 2007, which claims priority of U.S.Provisional Application No. 60/857,476, filed on Nov. 8, 2006, which arehereby incorporated by reference in their entirety. All patent andnon-patent references cited in this application are also herebyincorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to a method for immunizing an avianspecies with a plurality of antigens and generating a library comprisinga plurality of different antibodies raised in said avian species.

BACKGROUND OF INVENTION

Antibodies are widely used as therapeutic agents, diagnostic tools,research tools, and more.

Polyclonal antibodies are conventionally obtained by the time consumingmethod of immunizing a suitable host, usually a mammalian animal, with acomposition comprising the molecule of interest as an antigen and asuitable adjuvant. Several weeks later antibodies, generated by severalB cell clones, hence the term polyclonal, specific to the antigen can beharvested by bleeding the animal and collecting the serum. Polyclonalantibodies may bind different parts of the antigen although individualpolyclonal antibodies may bind virtually the same parts of an antigenwith different affinities.

Polyclonal antibodies are usually obtained from mammalian host animalssuch as rats, mice, goats, horses, sheep, rabbits, pigs and others. Alarge amount of experimental animals are used to generate differentantibodies—one animal is used per antigen. The animal will experiencediscomfort associated with the immunization and in particular thebleeding procedures. It is expensive and laborious to obtain antibodiesspecific to a large number of different antigens, since a similar largeamount of animals is required.

On one occasion however, the possibility of immunizing individualanimals with more than one antigen has been discussed in connection witha semi-automated method of hybridoma generation using mice immunizedwith multiple antigens and a novel antigen microarray assay (Proteomics5: 4070-4081, 2005 and WO 03089471). In this document, mice wereimmunized with 10 μg of 5-10 different protein antigens having sizesranging from about 12 to about 48 kDa in order to solve the bottleneckproblem in providing monoclonal antibodies to proteins encoded by thehuman genome. In the discussion of the scientific paper it is statedthat “It is also possible that increasing the number of immunogens foreach animal may ultimately lower the number of positive clones in anyone fusion. Indeed, subsequent production runs within our laboratoryhave shown that five antigens per animal are optimal”. A major problemassociated with immunizing with more than one antigen is, according toWO 03089471, immunodominance. In order to circumvent this problem, theanimal was injected with several antigen boosters. Immunization withmore than one antigen is not recommended, apart from perhaps in specialcases involving automated procedures for generating mice monoclonalhybridoma B cell lines, in which case it is recommended to immunize withno more than 5-10 different purified protein antigens. In the patentapplication it is also suggested that that immunization might be carriedout with up to 50 different antigens. However, no specific examplesdemonstrate that this approach will work with a reasonable expectationof success.

Conventionally obtained antibodies have several other drawbacks inaddition to being relatively expensive and time consuming to produce. Itmay e.g. be difficult to obtain a sufficient response toward mammalianantigens. In general, the large degree of conservation of immunoglobulinstructure, immunoglobulin receptor structures, etc. among mammals doesin many cases give rise to inaccurate test results when using mammalianantibodies for e.g. detecting mammalian antigens. Also, the problemswith protein A/protein G binding, interference with rheumatoid factor,and activation of mammalian complement are often encountered when usingmammalian antibodies and mammalian antigens.

In The FASEB Journal 4:2528-2532, 2001 it is disclosed that byimmunizing a chicken during a 20 day period with a total amount of about30-300 μg antigen, an immune response could be detected about 2-3 weekslater. It is concluded that the egg yolk is a convenient source ofpolyclonal antibodies because:

-   -   the quantity of antigen needed is much lower compared to        rabbits,    -   birds produce antibodies against highly conserved mammalian        antigens,    -   the use of complete Freunds adjuvant leads to very high and long        lasting titers of yolk antibodies, starting as early as 16 days        after the first immunization,    -   the purification of antibodies is simple and quick, and a purity        of 90% is easily attained by PEG precipitation,    -   chicken are inexpensive to keep and easy to handle,    -   chicken antibodies are acid- and heat-resistant and therefore        might be orally applied to prevent or to cure intestinal        diseases of young animals or humans.

The document does not however, discuss if specific antibodies can begenerated in a much faster and much more inexpensive way.

It is thus known that a number of advantages are associated withobtaining antibodies in a non-invasive way from eggs from an immunizedchicken rather than collecting blood from an immunized mammal. However,the time required for generating antibodies is not dramatically reducedwhen using hen/chicken antibodies. And also, a large amount of birds areused to generate antibodies in cases where a large amount of differentantigens are used. The available literature in this field does notprovide any suggestions for providing specific antibodies in adramatically faster and more inexpensive way.

In Handbook of Laboratory Animal Science, Second Edition, Vol 1, JannHau e.g. it is disclosed that “For each antigen, there is a dose calledthe “window of immunogenicity.” Too much or too little antigen mayinduce suppression, sensitization, tolerance, or other unwantedimmunomodulation⁷. Very low doses (<1 to 5 μg) are used to inducehypersensitivity (allergy) and should be avoided in immunization ofanimals, particularly because booster injections may result inanaphylactic shock in the animals.”

There exists a dogma within the field of immunology that repeatedimmunizations with low doses of allergen may result in anaphylacticshock and that it is not possible to immunize animals with a largeamount of antigens.

SUMMARY OF INVENTION

The invention as disclosed relates to a fast and inexpensive method ofgenerating a library of antibodies each of these being specific towardsa pool of antigens. The antibody library is preferably a polyclonalantibody library generated by immunization of a vertebrate animal with apool of antigens such as an antigen library. The vertebrate animal ispreferably a hen of the species Gallus gallus. The antigen library maycomprise a multitude of compounds, preferably peptides that are capableof raising an immunogenic response in the host animal in combinationwith the carriers and adjuvants with which the antigen library isco-immunized.

Antibodies have been raised in animals for decades—using one antigen peranimal. According to the present invention however, large numbers ofantigen specific antibodies can surprisingly be obtained by immunizingan animal with a large number of antigens. A scientific dogma and atechnical prejudice have been overcome by successfully immunizinganimals with a large number of antigens.

The invention further relates to a method of isolating antigen specificantibodies from an antibody library, wherein the method comprisesimmobilizing the antigen, contacting the antibody library with theimmobilized antigen, removing unbound antibody, and recovering antigenspecific antibodies.

Normally, affinity chromatography is used for capturing antigen by meansof immobilized antibodies. By reversing this principle, the possibilityof isolating antigen specific antibodies from an antibody libraryarises. It follows that the likelihood of isolating polyclonalantibodies specific to any antigen increases with increasing number ofdifferent antigens used for immunization. As a result, a tool forproviding specific polyclonal antibodies within a very short time isprovided by bypassing the steps of immunization and waiting for animmune response to occur.

Finally, the present invention relates to the products resulting fromthe various methods.

Definitions

Adjuvant: Any substance whose admixture with an administered immunogenicdeterminant/antigen increases or otherwise modifies the immune responseto said determinant. Immunisation of the animal may be carried out withadjuvants and/or pharmaceutical carriers. An adjuvant is any substancethat enhances the immune response to an antigen with which it is mixed.The antigen may also be mixed with two or more different adjuvants priorto immunisation. Examples of commonly used adjuvants comprise:Immunostimulatory oil emulsions (for example, water-in-oil,oil-in-water, water-in-oil-in-water such as e.g. Freund's incompleteadjuvant such as Montainde®, Specol, mineral salts such e.g. as Al(OH)₃,AIPO₄, microbial products, Saponins such as Qual A, synthetic products,as well as adjuvant formulations, and immune stimulatory complexes(ISCOMs). A list of other commonly used adjuvants is disclosed on pages6-8 in WO 03089471, the list being hereby incorporated by reference. Themost preferred adjuvant according to the present invention is Specolsince a fast immune response is initiated. Normally, antibodies can beharvested three to six weeks after immunisation in e.g. hen, but whenusing Specol as an adjuvant, antibodies may be harvested after onlyabout two weeks. Adjuvants are generally included in the immunogeniccompositions in an amount according to the instructions of themanufacturer. If the adjuvant is Specol an amount of e.g. 5, 10, 15, 25,50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000 μl may be added to the immunogenic composition.Preferably, the adjuvant is mixed with water or buffer in an amount ofabout 1:1.

Amino acid: Any synthetic or naturally occurring amino carboxylic acid,including any amino acid occurring in peptides and polypeptidesincluding proteins and enzymes synthesized in vivo. Natural amino acidscomprise an amino terminal part (NH₂) and a carboxy terminal part (COOH)separated by a central part comprising a carbon atom, or a chain ofcarbon atoms, comprising at least one side chain or functional group.NH₂ refers to the amino group present at the amino terminal end of anamino acid or peptide, and COOH refers to the carboxy group present atthe carboxy terminal end of an amino acid or peptide. The generic termamino acid comprises both natural and non-natural amino acids. Naturalamino acids of standard nomenclature as listed in J. Biol. Chem.,243:3552-59 (1969) and adopted in 37 C.F.R., section 1.822(b)(2) belongto the group of amino acids listed in Table 2 herein below. Non-naturalamino acids are those not listed in Table 2. Examples of non-naturalamino acids are those listed e.g. in 37 C.F.R. section 1.822(b)(4), allof which are incorporated herein by reference.

Further examples of non-natural amino acids are listed herein below.Amino acid residues described herein can be in the “D” or “L” isomericform.

TABLE 2 Natural amino acids and their respective codes. Symbols 1-Letter3-Letter Amino acid Y Tyr tyrosine G Gly glycine F Phe phenylalanine MMet methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucineT Thr threonine V Val valine P Pro proline K Lys lysine H His histidineQ Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine C Cys cysteine U Sec selenocysteine OPyl pyrrolysine

Amino acid residue: the term “amino acid residue” is meant to encompassamino acids, either standard amino acids, non-standard amino acids orpseudo-amino acids, which have been reacted with at least one otherspecies, such as 2, for example 3, such as more than 3 other species. Inparticular amino acid residues may comprise an acyl bond in place of afree carboxyl group and/or an amine-bond and/or amide bond in place of afree amine group. Furthermore, reacted amino acids residues may comprisean ester or thioester bond in place of an amide bond

Amino acid precursor: Moiety capable of generating an amino acid residuefollowing incorporation of the precursor into a peptide.

Antibody: Immunoglobulin molecule or immunologically active portionthereof, i.e. molecules that contain an “antigen binding site” orparatope. Immunoglobulins may be natural or wholly or partiallysynthetically produced. All fragments and derivatives thereof whichmaintain specific binding ability are also included in the term. Theterm also covers any polypeptide having a binding domain which ishomologous or largely homologous, such as at least 95% identical, to animmunoglobulin binding domain. These polypeptides may be derived fromnatural sources, or partly or wholly synthetically produced. An antibodymay be monoclonal or polyclonal. The antibody may be a member of anyimmunoglobulin class, including IgY, IgG, IgM, IgA, IgD, and IgE.Derivatives of the IgY class, however, are preferred in the presentinvention. An antibody that “specifically binds to” or is “specific for”a particular polypeptide or an epitope on a particular polypeptide isone that binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope. Antibodies can furthermore be described as plasmaproteins that bind specifically to particular molecules known asantigens. Antibody molecules are produced by B lymphocytes in responseto immunisation with antigen. They are the specific molecules of thehumoral immune response that bind to and neutralize pathogens or preparethem for uptake and destruction by phagocytes. Each antibody moleculehas a unique structure that allows it to bind its specific antigen, butall antibodies have the same overall structure and are knowncollectively as immunoglobulins. The body can make an almost unlimitedvariety of different antibodies, each B lymphocyte being geneticallyprogrammed early in its development to produce antibodies of a singlespecificity. A pool of antibodies according to the present invention canbe obtained by immunising a single animal with a randomly generatedpeptide library. A randomly generated peptide library will in generalstimulate an antibody response comprising antibodies with bindingcapability of (in theory) any antigen.

Antibody fragment: refers to any derivative of an antibody which is lessthan full-length. Preferably, the antibody fragment retains at least asignificant portion of the full-length antibody's specific bindingability. Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, scFv, Fv, dsFv diabody, and Fd fragments. Theantibody fragment may be produced by any means. For instance, theantibody fragment may be enzymatically or chemically produced byfragmentation of an intact antibody or it may be recombinantly producedfrom a gene encoding the partial antibody sequence. Alternatively, theantibody fragment may be wholly or partially synthetically produced. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multimolecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids.

Antibody specificity toward an antigen: According to the presentinvention an antibody can be termed antigen specific if e.g. antigen:antibody binding can be demonstrated in an assay as disclosed in Example4, where antigen is linked to a carrier in the presence of PBS andantibodies are likewise suspended in a PBS buffer.

According to the present invention, at least about 1% of the antibodiesobtained subsequent to immunisation show specificity to the antigensused for immunisation. If less than 1% of the antibodies showspecificity to the antigens, antibodies may e.g. have been harvested toosoon after immunisation, insufficient amounts of antigen have beenemployed, or inefficient amounts of adjuvants have been employed. Morepreferably, at least about 2, 3, 4, or 5% of the antibodies are antigensspecific. Most preferably, at least about 6, 7, 8, 9, or 10% of theantibodies are specific. And most preferably about 20-30% of theantibodies are antigen specific.

Antigen: Any substance or molecule that can react with or bind to aspecific antibody or specific antibodies. Conditions suitable for anantigen:antibody binding to take place are well known in the art. In theExamples, PBS buffer is employed.

Boost: To boost by a booster shot or dose is to give an additional doseof an immunizing agent, such as a vaccine, given at a time after theinitial dose to sustain the immune response elicited by the previousdose of the same agent.

Bursa of Fabricius/Bursa fabricii: A thymus-like specialized lymphoidgland in birds that is an outgrowth of the cloaca and the site ofhematopoiesis and B cell maturation in birds. It is thus the residenceof antibody producing B cells.

Conjugated: An association formed between an immunogenic determinant anda carrier. The association may be a physical association generated e.g.by the formation of a chemical bond, such as e.g. a covalent bond,formed between the immunogenic determinant and the carrier.

Carriers: Entity or compound to which antigens are coupled to aid in theinduction of an immune response. Conjugation to a carrier is importantbecause peptides are small molecules that alone do not tend to beimmunogenic, thus possibly eliciting a weak immune response. The carriercontains many epitopes that stimulate T-helper cells, which help inducethe B-cell response. Many different carriers can be used for coupling tosynthetic peptides. The most commonly selected carriers are keyholelimpet hemacyanin (KLH) and bovine serum albumin (BSA). The higherimmunogenicity of KLH often makes it the preferred choice. Anotheradvantage of choosing KLH over BSA is that BSA is used as a blockingagent in many experimental assays. Because antisera raised againstpeptides conjugated to BSA will also contain antibodies to BSA, falsepositives may result. Although KLH is large and immunogenic, it mayprecipitate during cross-linking, making it difficult to handle in somecases. Ovalbumin (OVA) is another useful carrier protein. It is a goodchoice as a second carrier protein when verifying whether antibodies arespecific for the peptide alone and not the carrier. Rabbit Serum Albumin(RSA) may be used when the antibody response to the carrier protein mustbe kept to a minimum. Rabbits immunised with RSA conjugate are lesslikely to raise antibodies to the carrier, as the RSA is recognized as“self.” If the RSA conjugate were injected into another host, theprotein would not be recognized as self. The same principle, of course,applies when immunising with chicken-derived carriers in chicken. It isimportant to recognize that the immune system reacts to thepeptide-protein carrier as a whole and that there will be a portion ofresponse directed against the conjugated peptide as well as the linkerand the carrier. When screening by ELISA it is advisable to use apeptide conjugate prepared using a different carrier. This is notnecessary if performing ELISA assays where the plates are coateddirectly with unconjugated peptide. Suitable carriers will thus be ofprotein origin in most cases. In some cases however, the carrier may beof non-protein origin such as e.g. polymers comprising carbohydrates,etc. It follows that it may not be necessary to conjugate relativelylarge peptides with a carrier in order to obtain an immune response.Peptides with a length of at least about 100 amino acids and morepreferably with a length of at least about 150 amino acids may not needcarrier conjugation in order to function as immunogens in the presentinvention. It is however advisable to conjugate all peptides withcarrier molecules without regard to the size. Immunisation of the animalmay be carried out with or without pharmaceutical carriers. Suitablecarriers are typically large, slowly metabolized macromolecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates (such asoil droplets or liposomes), and inactive virus particles. Such carriersare well known to those of ordinary skill in the art. Additionally,these carriers may function as immunostimulating agents (“adjuvants”).See definition section “Adjuvant”.

Chemistry:

The term “hydrido” denotes a single hydrogen atom (H). This hydridoradical may be attached, for example, to an oxygen atom to form ahydroxyl radical or two hydrido radicals may be attached to a carbonatom to form a methylene (—CH₂—) radical.

Where the term “alkyl” is used, either alone or within other terms suchas “haloalkyl” and “alkylsulfonyl”, it embraces linear or branchedradicals having one to about twenty carbon atoms or, preferably, one toabout twelve carbon atoms. Preferred alkyl radicals are “lower alkyl”radicals having one to about ten carbon atoms, such as lower alkylradicals having one to about six carbon atoms. Examples of such radicalsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like. Branchedchain isomers of straight chain alkyl groups, include, but are notlimited to, the following which are provided by way of example:—CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃, —C(CH₂CH₃)₃,—CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃,—CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. When substituted, the“alkyl” or “lower alkyl” can comprise one or more radicals selected fromthe group of radicals consisting of hydroxy, primary amine, carboxy,acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, andthiolyl.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twenty carbon atoms, suchas from two to about twelve carbon atoms, for example from two to abouteight carbon atoms. Preferred alkyl radicals are “lower alkenyl”radicals having two to about six carbon atoms. Examples of such radicalsinclude ethenyl, n-propenyl, butenyl, and the like. When substituted,the “alkenyl” or “lower alkenyl” can comprise one or more radicalsselected from the group of radicals consisting of hydroxy, primaryamine, carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro,cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms. The term “haloalkyl” embraces radicals wherein any one ormore of the alkyl carbon atoms is substituted with halo as definedabove. Specifically embraced are monohaloalkyl, dihaloalkyl andpolyhaloalkyl radicals. A monohaloalkyl radical, for one example, mayhave either an iodo, bromo, chloro or fluoro atom within the radical.Dihalo and polyhaloalkyl radicals may have two or more of the same haloatoms or a combination of different halo radicals. “Lower haloalkyl”preferably embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,trichloromethyl, pentafluoroethyl, heptafluoropropyl,difluorochloromethyl, dichlorofluoromethyl, difluoroethyl,difluoropropyl, dichloroethyl and dichloropropyl. The “haloalkyl” or“lower haloalkyl” can optionally be further substituted. When furthersubstituted, the “haloalkyl” or “lower haloalkyl” can further compriseone or more radicals selected from the group of radicals consisting ofhydroxy, primary amine, carboxy, acid chloride, sulfonyl chloride,sulphonate, nitro, cyano, isothiocyanate, phosphonyl, sulphonyl,sulfamyl, carbonyl, and thiolyl.

The term “hydroxyalkyl” embraces linear or branched alkyl radicalshaving from one to about ten carbon atoms any one of which may besubstituted with one or more hydroxyl radicals. Hydroxyalkyl radicalscan be “lower hydroxyalkyl” radicals preferably having one to six carbonatoms and one or more hydroxyl radicals. Examples of such radicalsinclude hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl andhydroxyhexyl. The “hydroxyalkyl” or “lower hydroxyalkyl” can optionallybe further substituted. When further substituted, the “hydroxyalkyl” or“lower hydroxyalkyl” can further comprise one or more radicals selectedfrom the group of radicals consisting of primary amine, carboxy, acidchloride, sulfonyl chloride, sulphonate, nitro, cyano, isothiocyanate,halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl.

The terms “alkoxy” and “alkoxyalkyl” embrace linear or branchedoxy-containing radicals each having alkyl portions of one to about tencarbon atoms, such as methoxy radical. Alkoxy radicals can be “loweralkoxy” radicals having one to six carbon atoms. Examples of suchradicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.

The term “alkoxyalkyl” also embraces alkyl radicals having two or morealkoxy radicals attached to the alkyl radical, that is, to formmonoalkoxyalkyl and dialkoxyalkyl radicals. Alkoxyalkyl radicals can be“lower alkoxyalkyl” radicals having one to six carbon atoms and one ortwo alkoxy radicals. Examples of such radicals include methoxymethyl,methoxyethyl, ethoxyethyl, methoxybutyl and metoxypropyl. The alkyl insaid “alkoxyalkyl” can be substituted with one or more of hydroxy,primary amine, carboxy, acid chloride, sulfonyl chloride, sulphonate,nitro, cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl. When e.g. the above “alkoxyl” or “alkoxyalkyl”radicals are substituted with one or more halo atoms, such as fluoro,chloro or bromo, “haloalkoxy” or “haloalkoxyalkyl” radicals areprovided. Examples of such radicals include fluoromethoxy,chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy andfluoropropoxy.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one, two or three rings wherein such rings may beattached together in a pendent manner or may be fused. When substituted,“aryl” can comprise one or more radicals selected from the group ofradicals consisting of hydroxy, primary amine, carboxy, acid chloride,sulfonyl chloride, sulphonate, nitro, cyano, isothiocyanate, halogen,phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl. Examples of“aryl” include aromatic radicals such as phenyl, pentafluorphenyl,naphthyl, tetrahydronaphthyl, indane and biphenyl.

The term “heterocyclic” embraces saturated, partially saturated andunsaturated heteroatom-containing ring-shaped radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen. Whensubstituted, “heterocyclic” can comprise one or more radicals selectedfrom the group of radicals consisting of hydroxy, primary amine,carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, andthiolyl. Examples of saturated heterocyclic radicals include e.g.saturated 3 to 6-membered heteromonocylic group containing 1 to 4nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidino,piperazinyl, etc.]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl, etc.]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.thiazolidinyl, etc.]. Examples of partially saturated heterocyclicradicals include dihydrothiophene, dihydropyran, dihydrofuran anddihydrothiazole.

The term “heteroaryl” embraces unsaturated heterocyclic radicals. Whensubstituted, “heteroaryl” can comprise one or more radicals selectedfrom the group of radicals consisting of hydroxy, primary amine,secondary amine, carboxy, acid chloride, sulfonyl chloride, sulphonate,nitro, cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl. Examples of unsaturated heterocyclic radicals,also termed “heteroaryl” radicals, include e.g. unsaturated 5 to 6membered heteromonocyclic group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl 2-pyridyl,3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl[e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.]tetrazolyl [e.g. 1H-tetrazolyl, 2H-tetrazolyl, etc.], etc.; unsaturatedcondensed heterocyclic group containing 1 to 5 nitrogen atoms, forexample, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl,isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g.,tetrazolo[1,5-b]pyridazinyl, etc.], etc.; unsaturated 3 to 6-memberedheteromonocyclic group containing an oxygen atom, for example, pyranyl,2-furyl, 3-furyl, etc.; unsaturated 5 to 6-membered heteromonocyclicgroup containing a sulfur atom, for example, 2-thienyl, 3-thienyl, etc.;unsaturated 5- to 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl,isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,5-oxadiazolyl, etc.] etc.; unsaturated condensed heterocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.benzoxazolyl, benzoxadiazolyl, etc.]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.] etc.;unsaturated condensed heterocyclic group containing 1 to 2 sulfur atomsand 1 to 3 nitrogen atoms [e.g., benzothiazolyl, benzothiadiazolyl,etc.] and the like. The term “heteroaryl” or “unsaturated heterocyclicradical” also embraces radicals where heterocyclic radicals are fusedwith aryl radicals. Examples of such fused bicyclic radicals includebenzofuran, benzothiophene, and the like. Said “heterocyclic group” canbe substituted with one or more radicals selected from the group ofradicals consisting of hydroxy, primary amine, carboxy, acid chloride,sulfonyl chloride, sulphonate, nitro, cyano, isothiocyanate, halogen,phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl, saidsubstitution generating a substituted “heteroaryl”, optionally asubstituted “heteroaryl” fused with an “aryl” radical which can besubstituted or un-substituted. When substituted, the “aryl” issubstituted as described herein above. Preferred heterocyclic radicalsinclude five to ten membered fused or unfused radicals. More preferredexamples or heteroaryl radicals include benzofuryl,2,3-dihydrobenzofuryl, benzotrienyl, indolyl, dihydroindolyl, chromanyl,benzopyran, thiochromanyl, benzothiopyran, benzodioxolyl, benzodioxanyl,pyridyl, thienyl, thiazolyl, oxazolyl, furyl, and pyrazinyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

“Alkylsulfonyl” embraces alkyl radicals attached to a sulfonyl radical,where alkyl can be substituted is defined as above. Alkylsulfonylradicals can be “lower alkylsulfonyl” radicals having one to six carbonatoms. Examples of such lower alkylsulfonyl radicals includemethylsulfonyl, ethylsulfonyl and propylsulfonyl.

The term “arylsulfonyl” embraces aryl radicals as defined above,including substituted aryl radicals, attached to a sulfonyl radical.Examples of such radicals include phenylsulfonyl.

The terms “sulfamyl,” “aminosulfonyl” and “sulfonamidyl,” whether aloneor used with terms such as “N-alkylaminosulfonyl”,“N-arylaminosulfonyl”, “N,N-dialkylaminosulfonyl” and“N-alkyl-N-arylaminosulfonyl”, denotes a sulfonyl radical substitutedwith an amine radical, forming a sulfonamide (—SO₂NH₂).

The terms “N-alkylaminosulfonyl” and “N,N-dialkylaminosulfonyl” denotesulfamyl radicals substituted respectively, with one alkyl radical, ortwo alkyl radicals, optionally substituted alkyl radicals as describedherein above. Alkylaminosulfonyl radicals can be “loweralkylaminosulfonyl” radicals having one to six carbon atoms. Examples ofsuch lower alkylaminosulfonyl radicals include N-methylaminosulfonyl,N-ethylaminosulfonyl and N-methyl-N-ethylaminosulfonyl.

The terms “N-arylaminosulfonyl” and “N-alkyl-N-arylaminosulfonyl” denotesulfamyl radicals substituted, respectively, with one aryl radical, orone alkyl and one aryl radical, optionally substituted aryl and/or alkylradicals as described herein above. N-alkyl-N-arylaminosulfonyl radicalscan be “lower N-alkyl-N-arylsulfonyl” radicals having alkyl radicals ofone to six carbon atoms. Examples of such lower N-alkyl-N-arylaminosulfonyl radicals include N-methyl-phenylaminosulfonyl andN-ethyl-phenylaminosulfonyl.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —CO₂H.

The term “carboxyalkyl” or “alkanoyl” embraces radicals having a carboxyradical as defined above, attached to an alkyl radical as describedherein above. When substituted, the “alkyl” or “lower alkyl” cancomprise one or more radicals selected from the group of radicalsconsisting of hydroxy, primary amine, carboxy, acid chloride, sulfonylchloride, sulphonate, nitro, cyano, isothiocyanate, halogen, phosphonyl,sulphonyl, sulfamyl, carbonyl, and thiolyl. Examples of “carboxyalkyl”radicals include formyl, acetyl, propionyl (propanoyl), butanoyl(butyryl), isobutanoyl (isobutyryl), valeryl (pentanoyl), isovaleryl,pivaloyl, hexanoyl or the like.

The term “carbonyl”, whether used alone or with other terms, such as“alkylcarbonyl”, denotes —(C═O)—.

The term “alkylcarbonyl” embraces radicals having a carbonyl radicalsubstituted with an alkyl radical. Alkylcarbonyl radicals can be “loweralkylcarbonyl” radicals having from one to six carbon atoms. Examples ofsuch radicals include methylcarbonyl and ethylcarbonyl. Whensubstituted, the “alkyl” or “lower alkyl” of the “alkylcarbonyl” cancomprise one or more radicals selected from the group of radicalsconsisting of hydroxy, primary amine, carboxy, acid chloride, sulfonylchloride, sulphonate, nitro, cyano, isothiocyanate, halogen, phosphonyl,sulphonyl, sulfamyl, and thiolyl.

The term “alkylcarbonylalkyl”, denotes an alkyl radical substituted withan “alkylcarbonyl” radical as described herein above. Both the alkyl andthe alkylcarbonyl can be substituted as described herein above.

The term “alkoxycarbonyl” means a radical containing an alkoxy radical,as defined above, attached via an oxygen atom to a carbonyl radical.“Lower alkoxycarbonyl” embraces alkoxy radicals preferably having fromone to six carbon atoms. Examples of “lower alkoxycarbonyl” esterradicals include substituted or unsubstituted methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl and hexyloxycarbonyl.

The term “alkoxycarbonylalkyl” embraces radicals having“alkoxycarbonyl”, as defined above substituted to an optionallysubstituted alkyl radical. Alkoxycarbonylalkyl radicals can be “loweralkoxycarbonylalkyl” having lower alkoxycarbonyl radicals as definedabove attached to one to six carbon atoms. Examples of such loweralkoxycarbonylalkyl radicals include methoxycarbonylm ethyl,tert-butoxycarbonylethyl, and methoxycarbonylethyl.

The term “aminocarbonyl” when used by itself or with other terms such as“aminocarbonylalkyl”, “N-alkylaminocarbonyl”, “N-arylaminocarbonyl,“N,N-dialkylaminocarbonyl”, “N-alkyl-N-arylaminocarbonyl”,“N-alkyl-N-hydroxyaminocarbonyl” and“N-alkyl-N-hydroxyaminocarbonylalkyl”, denotes an amide group of theformula —C(═O)NH₂.

The terms “N-alkylaminocarbonyl” and “N,N-dialkylaminocarbonyl” denoteaminocarbonyl radicals which have been substituted with one alkylradical and with two alkyl radicals, respectively. The alkyl radicalscan be substituted as described herein above. “Lower alkylaminocarbonyl”comprises lower alkyl radicals as described above attached to anaminocarbonyl radical.

The terms “N-arylaminocarbonyl” and “N-alkyl-N-arylaminocarbonyl” denoteaminocarbonyl radicals substituted, respectively, with one aryl radical,or one alkyl and one aryl radical, wherein such radicals can besubstituted as described herein above.

The term “aminocarbonylalkyl” embraces optionally substituted alkylradicals substituted with aminocarbonyl radicals.

The term “N-cycloalkylaminocarbonyl” denotes aminocarbonyl radicalswhich have been substituted with at least one optionally substitutedcycloalkyl radical. “Lower cycloalkylaminocarbonyl” comprises lowercycloalkyl radicals of three to seven carbon atoms, attached to anaminocarbonyl radical.

The term “aminoalkyl” embraces alkyl radicals substituted with one ormore amino radicals. The alkyl radicals can be further substituted byone or more radicals selected from the group of radicals consisting ofhydroxy, carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro,cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl.

The term “alkylaminoalkyl” embraces aminoalkyl radicals having thenitrogen atom substituted with an optionally substituted alkyl radical.

The term “amidino” denotes an —C(═NH)—NH₂ radical.

The term “cyanoamidino” denotes an —C(═N—CN)—NH₂ radical.

The term “heterocyclicalkyl” embraces heterocyclic-substituted alkylradicals. The alkyl radicals can themselves be substituted by one ormore radicals selected from the group of radicals consisting of hydroxy,primary amino, carboxy, acid chloride, sulfonyl chloride, sulphonate,nitro, cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl. Heterocyclicalkyl radicals can be “lowerheterocyclicalkyl” radicals preferably having from one to six carbonatoms and a heterocyclic radical. Examples include such radicals aspyrrolidinylmethyl, pyridylmethyl and thienylmethyl.

The term “aralkyl” embraces aryl-substituted alkyl radicals. The alkylradicals can themselves be substituted by one or more radicals selectedfrom the group of radicals consisting of hydroxy, primary amino,carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, andthiolyl. Aralkyl radicals can be “lower aralkyl” radicals having arylradicals attached to alkyl radicals having from one to six carbon atoms.Examples of such radicals include benzyl, diphenylmethyl,triphenylmethyl, phenylethyl and diphenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy. The terms benzyl and phenylmethyl are interchangeable.

The term “cycloalkyl” embraces radicals having three to ten carbonatoms. Cycloalkyl radicals can be “lower cycloalkyl” radicals havingthree to seven carbon atoms. Examples include radicals such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The“cycloalkyl” can optionally be substituted by one or more radicalsselected from the group of radicals consisting of hydroxy, primaryamine, carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro,cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl.

The term “cycloalkenyl” embraces unsaturated cyclic radicals havingthree to ten carbon atoms. The “cycloalkenyl” can optionally besubstituted by one or more radicals selected from the group of radicalsconsisting of hydroxy, primary amine, carboxy, acid chloride, sulfonylchloride, sulphonate, nitro, cyano, isothiocyanate, halogen, phosphonyl,sulphonyl, sulfamyl, carbonyl, and thiolyl. Examples includecyclobutenyl, cyclopentenyl, cyclohexenyl and cycloheptenyl, which canoptionally be substituted as described above.

The term “alkylthio” embraces radicals containing a linear or branchedalkyl radical, of one to ten carbon atoms, attached to a divalent sulfuratom. An example of “alkylthio” is methylthio, (CH₃—S—). The alkylradical can be substituted as described herein above.

The term “alkylsulfinyl” embraces radicals containing a linear orbranched alkyl radical, of one to ten carbon atoms, attached to adivalent —S(═O)— atom. The alkyl radical can be substituted as describedherein above.

The term “aminoalkyl” embraces alkyl radicals substituted with aminoradicals. The alkyl radicals can be further substituted by one or moreradicals selected from the group of radicals consisting of hydroxy,carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, andthiolyl. Aminoalkyl radicals can be “lower aminoalkyl” having from oneto six carbon atoms. Examples include aminomethyl, aminoethyl andaminobutyl which can optionally be further substituted as describedabove.

The term “alkylaminoalkyl” embraces aminoalkyl radicals having thenitrogen atom substituted with at least one alkyl radical.Alkylaminoalkyl radicals can be “lower alkylaminoalkyl” having one tosix carbon atoms attached to a lower aminoalkyl radical as describedabove. The alkyl radical can be substituted as described herein above.

The terms “N-alkylamino” and “N,N-dialkylamino” denote amino groupswhich have been substituted with one alkyl radical and with two alkylradicals, respectively. The alkyl radical can be substituted asdescribed herein above. Alkylamino radicals can be “lower alkylamino”radicals having one or two alkyl radicals of one to six carbon atoms,attached to a nitrogen atom. Suitable “alkylamino” may be mono ordialkylamino such as N-methylamino, N-ethylamino, N,N-dimethylamino,N,N-diethylamino or the like.

The term “arylamino” denotes amino groups which have been substitutedwith one or two aryl radicals, such as N-phenylamino. The “arylamino”radicals may be further substituted on the aryl ring portion of theradical. Substitutions can include one or more of hydroxy, amino,carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, andthiolyl.

The term “aralkylamino” denotes amino groups which have been substitutedwith one or two aralkyl radicals, such as N-benzylamino. The“aralkylamino” radicals may be further substituted on the aryl ringportion of the radical. Substitutions can include one or more ofhydroxy, amino, carboxy, acid chloride, sulfonyl chloride, sulphonate,nitro, cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl.

The terms “N-alkyl-N-arylamino” and “N-aralkyl-N-alkylamino” denoteamino groups which have been substituted with one aralkyl and one alkylradical, or one aryl and one alkyl radical, respectively, to an aminogroup. The aralkyl and/or alkyl and/or aryl radicals can be substitutedas described herein above.

The terms “N-arylaminoalkyl” and “N-aralkylaminoalkyl” denote aminogroups which have been substituted with one aryl radicals or one aralkylradical, respectively, and having the amino group attached to an alkylradical. The aralkyl and/or alkyl and/or aryl radicals can besubstituted as described herein above. Arylaminoalkyl radicals can be“lower arylaminoalkyl” having the arylamino radical attached to one tosix carbon atoms. Examples of such radicals include N-phenylaminomethyland N-phenyl-N-methylaminomethyl.

The terms “N-alkyl-N-arylaminoalkyl”, and “N-aralkyl-N-alkylaminoalkyl”denote N-alkyl-N-arylamino and N-alkyl-N-aralkylamino groups,respectively, and having the amino group attached to alkyl radicalswhich can be substituted as described herein above.

The term “acyl”, whether used alone, or within a term such as“acylamino”, denotes a radical provided by the residue after removal ofhydroxyl from an organic acid.

The term “acylamino” embraces an amino radical substituted with an acylgroup. An examples of an “acylamino” radical is acetylamino or acetamido(CH₃C(═O)—NH—) where the amine may be further substituted with alkyl,aryl or aralkyl, wherein said alkyl, aryl or aralkyl can be substitutedas described herein above.

The term “arylthio” embraces aryl radicals of six to ten carbon atoms,attached to a divalent sulfur atom. The aryl can be substituted asdescribed herein above. An example of “arylthio” is phenylthio.

The term “aralkylthio” embraces aralkyl radicals as described above,attached to a divalent sulfur atom. The aralkyl radicals can be furthersubstituted as described herein above. An example of “aralkylthio” isbenzylthio.

The term “aryloxy” embraces aryl radicals, as defined above, attached toan oxygen atom. The aryl can be substituted as described herein above.Examples of such radicals include phenoxy.

The term “aralkoxy” embraces oxy-containing aralkyl radicals attachedthrough an oxygen atom to other radicals. The aralkyl can be substitutedas described herein above. Aralkoxy radicals can be “lower aralkoxy”radicals having phenyl radicals attached to lower alkoxy radical asdescribed above.

The term “haloaralkyl” embraces aryl radicals as defined above attachedto haloalkyl radicals. The aryl can be further substituted as describedherein above.

The term “carboxyhaloalkyl” embraces carboxyalkyl radicals as definedabove having halo radicals attached to the alkyl portion. The alkylportion can be further substituted as described herein above.

The term “alkoxycarbonylhaloalkyl” embraces alkoxycarbonyl radicals asdefined above substituted on a haloalkyl radical. The haloalkyl radicalcan be further substituted by one or more of hydroxy, amino, carboxy,acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl.

The term “aminocarbonylhaloalkyl” embraces aminocarbonyl radicals asdefined above substituted on an optionally substituted haloalkyl radicalwherein the alkyl is substituted by one or more of hydroxy, amino,carboxy, acid chloride, sulfonyl chloride, sulphonate, nitro, cyano,isothiocyanate, phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl.

The term “alkylaminocarbonylhaloalkyl” embraces alkylaminocarbonylradicals as defined above substituted on an optionally substitutedhaloalkyl radical as described above.

The term “alkoxycarbonylcyanoalkenyl” embraces alkoxycarbonyl radicalsas defined above, and a cyano radical, both substituted on an optionallysubstituted alkenyl radical.

The term “carboxyalkylaminocarbonyl” embraces aminocarbonyl radicalssubstituted with carboxyalkyl radicals, as defined above. Thecarboxyalkyl can be further substituted. Substitutions can include oneor more of hydroxy, amino, acid chloride, sulfonyl chloride, sulphonate,nitro, cyano, isothiocyanate, halogen, phosphonyl, sulphonyl, sulfamyl,carbonyl, and thiolyl.

The term “aralkoxycarbonylalkylaminocarbonyl” embraces aminocarbonylradicals substituted with aryl-substituted alkoxycarbonyl radicals, asdefined above.

The term “cycloalkylalkyl” embraces cycloalkyl radicals having three toten carbon atoms attached to an alkyl radical, as defined above.Cycloalkylalkyl radicals can be “lower cycloalkylalkyl” radicals havingcycloalkyl radicals attached to lower alkyl radicals as defined above.Examples include radicals such as cyclopropylmethyl, cyclobutylmethyl,and cyclohexylethyl.

The term “aralkenyl” embraces optionally substituted aryl radicalsattached to alkenyl radicals having two to ten carbon atoms, such asphenylbutenyl, and phenylethenyl or styryl. When substituted the arylcan be substituted with one or more of hydroxy, amino, carboxy, acidchloride, sulfonyl chloride, sulphonate, nitro, cyano, isothiocyanate,halogen, phosphonyl, sulphonyl, sulfamyl, carbonyl, and thiolyl.

Co-immunization: Immunization by means of separate and/or sequentialadministration to an individual of an immunogenic determinant and acarrier.

“Conditions suitable for binding”: means those conditions (in terms ofsalt concentration, pH, detergent, polypeptide concentration,temperature, etc.) which allow for binding to occur between a ligand andits binding partner in solution i.e. the bidning between an antigen andan antibody. Preferably, the conditions are not so lenient that asignificant amount of nonspecific binding occurs.

Derivative: refers to polypeptides derived from naturally occurring XXXby chemical modifications such as ubiquitination, labeling (e.g., withradionuclides, various enzymes, etc.), pegylation (derivatization withpolyethylene glycol), or by insertion (or substitution by chemicalsynthesis) of amino acids (amino acids) such as ornithine, which do notnormally occur in human proteins.

Elution: By using different elution conditions, the method allows anapproximate quantification of the binding affinity of the monoclonalantibody for its binding partner. Elution agents that may be usedinclude chaotropic agents such as guanidine hydrochloride or urea atconcentrations between 10 μM and 8 M or ethylene glycol in an aqueoussolution of 0.01% to 100% w/v. Elutions may also be carried out usingaqueous or non-aqueous solutions of glycine. Elutions may be carried outusing aqueous or non aqueous solutions of triethylamine between 1 μM anda saturated solution, preferably 100 mM, at a pH of between pH 8 and pH13, preferably pH 11.5.

Epitope: A specific site on a protein to which only certain antibodiesbind.

Galliformes: The order of birds that includes grouse, ptarmigan,capercaillie, partridges, pheasants, quails, turkeys and peacocks. Theseare mainly grain-eating, heavy-bodied, ground-nesting birds, capable ofonly short, rapid flights. The cocks are usually more colourful than thehens. A preferred bird according to the present invention is a hen(Gallus gallus domestica) either in the adult stage or as a chicken atthe age of at least 17-19 weeks.

Host: as used herein is any species immunized and used for theproduction of an antibody library according to the present invention.The term includes any animal, mammalian or vertebrate. Herein thepreferred host is an avian species and preferably a female of thespecies.

Immunization: Process of inducing an immunological response in anorganism by the introduction of an antigen into the animal by anysuitable means. Preferably, the method of introduction involvesinjection. The animals may be immunised with the antigensintrasplenically, intravenously, intraperitoneally, intradermal^ orsubcutaneously or by any other suitable means. For practical andeconomical reasons, chickens kept under field conditions are vaccinatedintramusculary in the breast muscle. In the laboratory, chickens canalso be vaccinated subcutaneously in the neck.

The total number of immunisations will depend upon the type and dose ofthe antigen, as well as on the particular adjuvant employed. In apreferred embodiment, at least two immunisations should be given. If theantibody titres begin to decrease, booster immunisations can be given.The total amount of peptide employed for each immunisation may likewisevary. Amounts of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 μg arepreferably used in a single immunisation. But amounts of about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250,or 500 mg may also be used in a single immunisation.

Immunological response: Response to an immunogenic compositioncomprising an immunogenic determinant. An immune response involves thedevelopment in the host of a cellular- and/or antibody-mediated responseto the administered composition or vaccine in question. An immuneresponse generally involves the action of one or more of i) theantibodies raised, ii) B cells, iii) helper T cells, iv) suppressor Tcells, and v) cytotoxic T cells, directed specifically to an immunogenicdeterminant present in an administered immunogenic composition.

Immunogenic composition: Composition capable of raising an immunologicalresponse in an individual.

Immunogenic: Functionality associated with an entity capable ofeliciting an immunological response.

Immunostimulating effect: Functionality associated with an entitycapable of eliciting an enhanced immune response. An enhanced immuneresponse will be understood within the meaning of the observeddifference in the immune response measured as an enhancement of anantibody production and/or a cytotoxic T-cell activity, or otherwiseregistered, when an immunogenic composition is administered in thepresence or absence, respectively, of the entity. An immunogeniccomposition comprising the entity will be understood as being acomposition according to the present invention.

Individual: Any species or subspecies of bird, mammal, fish, amphibian,or reptile.

Isolated: used in connection with, polypeptides, and antibodiesdisclosed herein ‘isolated’ refers to these having been identified andseparated and/or recovered from a component of their natural, typicallycellular, environment. Nucleic acids, polypeptides, and antibodies ofthe invention are preferably isolated, and vaccines and othercompositions of the invention preferably comprise isolated nucleicacids, polypeptides or isolated antibodies.

Isolated polypeptide: a protein, or a variant or fragment thereof, whichconstitutes 90% or more of the protein contents of a given preparationas evaluated by standard methods known in the art of protein chemistry.

A polypeptide “fragment”, “portion”, or “segment”: is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, such as atleast about 17 or more amino acids in various embodiments. To be active,any polypeptide, peptide or polypeptide fragment must have sufficientlength to display biologic and/or immunologic activity on their own, orwhen conjugated to a carrier.

Library: a plurality of entities such as antigens or antibodies

Ligand: In biochemistry, a ligand is an effector, a molecule that bindsto a site on a macromolecule's surface by intermolecular forces, therebychanging the chemical conformation of the macromolecule. Once amolecule's conformation has changed, its ability to function in otherchemical reactions is altered. This binding is usually a reversiblereaction, i.e. it can be undone. Actual coordinate covalent bondsbetween a ligand and its target molecule are rare in biological systems.Ligands include substrates, inhibitors, activators, andneurotransmitters. Herein an antigen is a ligand to an antibody.

Monoclonal antibody: (mAB) A single type of antibody that is directedagainst a specific epitope (antigen, antigenic determinant) and isproduced by a single clone of B cells or a single hybridoma cell line,which is formed by the fusion of a lymphocyte cell with a myeloma cell.Some myeloma cells synthesize single antibodies naturally.

Non-natural amino acid: Any amino acid not included in Table 2 hereinabove. Non-natural amino acids include, but are not limited to modifiedamino acids, pseudo-amino acids, L-amino acids, and stereoisomers ofD-amino acids.

Non-standard amino acid: a non-standard amino acid is capable of beingincorporated into a peptide or peptide like structure by translationmediated by a ribosome. A non-standard amino acid according to thepresent invention is any amino acid comprising an amino group and acarboxyl group separated by an α-carbon. The amino acid may for examplebe selected from the group consisting of, Aib, NaI, Sar, Orn, Lysineanalogues DAP and DAPA or any of the amino acids described in U.S. Pat.No. 5,573,905. Furthermore, non-standard amino acids may be any of theabove mentioned or any standard amino acids which further comprises oneor more moieties selected from the group consisting of hydroxyl, bromo,fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocycle,aryl, heteroaryl, carboxyl, carboalkoyl, alkyl, alkenyl, nitro, amino,alkoxyl and/or amido. The non-standard amino acid is capable of beingincorporated into a peptide or peptide like structure by translationmediated by a wt, mutant, modified or recombinant ribosome.

Normal physiological condition: means conditions that are typical insidea living organism or a cell. While it is recognized that some organs ororganisms provide extreme conditions, the intra-organismal andintracellular environment normally varies around pH 7 (i.e., from pH 6.5to pH 7.5), contains water as the predominant solvent, and exists at atemperature above 0° C. and below 50° C. It will be recognized that theconcentration of various salts depends on the organ, organism, cell, orcellular compartment used as a reference.

Nucleic acid: A chain or sequence of nucleotides that convey geneticinformation. In regards to the present invention the nucleic acid is adeoxyribonucleic acid (DNA).

Nucleic acid construct: A genetically engineered nucleic acid. A(nucleic acid) construct typically comprises several elements such asgenes or fragments of same, promoters, enhancers, terminators, polyAtails, linkers, markers or others.

Peptide: Plurality of covalently linked amino acid residues defining asequence and linked by amide bonds. The term is used analogously witholigopeptide and polypeptide. The amino acids may be both natural aminoacids and non-natural amino acids, including any combination thereof.The natural and/or non-natural amino acids may be linked by peptidebonds or by non-peptide bonds. The term peptide also embracespost-translational modifications introduced by chemical orenzyme-catalyzed reactions, as are known in the art. Suchpost-translational modifications can be introduced prior topartitioning, if desired. Amino acids as specified herein willpreferentially be in the L-stereoisomeric form. Amino acid analogs canbe employed instead of the 20 naturally-occurring amino acids. Severalsuch analogs are known, including fluorophenylalanine, norleucine,azetidine-2-carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophanand the like.

Peptide antigen: Peptides according to the present invention are definedas peptides with a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids. Peptides according to the presentinvention may also have lengths of about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600 amino acidsor more. The peptides used as antigens according to the invention can bea mix of peptides of different lengths, i.e. there may be short peptidespresent with a length as short as 6 amino acids as well as longerpeptides. Some or all of the peptides may also be in form of proteinsbeing either denatured or in their naturally folded form. It followsthat proteins can be obtained from either a natural source or they canbe recombinantly produced and subsequently purified. The peptides may inaddition comprise any form of modifications such as e.g. glycosylation,lipid attachment, hydroxylation, disulfide bonding, phosphorylation,etc. The peptides may also originate from a proteolytical digest ofpeptides and/or proteins. Antigens according to the present inventionare in other words of peptide origin with a length at or above 6 aminoacids, and may be derived either from natural sources, produced byrecombinant methods, or synthesized artificially—or any mixturesthereof. It is however important that the peptides used for immunisationin connection with the present invention are substantially free frome.g. contaminating cellular debris. Thus, the present invention does notrelate to immunisation with antigens derived from cellular materialincluding cell membranes, nucleic acids, etc. The amount of differentpeptide antigens used for immunisation of the animal is at least 15. Asthe aim of the present invention is to provide tools for rapidlyisolating antigen specific antibodies, it is advantageous to immunisewith even more antigens, as the likelihood thus increases of obtainingantibodies with specificity to most antigens. It is therefore preferableto immunise with at least about 25, 50, 100, 150, 200, 250, 500, 1000,5000, 10000, 50000, 100000, 500000, 1000000, or 5000000 differentantigens. It is most preferable to immunise with a syntheticallysynthesized peptide library comprising, in theory, about 20⁶ or moredifferent peptide combinations.

Peptide library: A preferred way of carrying out the present inventionis to synthesize a peptide library with peptides of similar lengthscovering the possible amino acid combinations of the 20 naturallyoccurring amino acids. Peptide synthesis may take place in solution oron a solid phase. A peptide library with peptides of a length of e.g. 6naturally occurring amino acids will thus in theory have 20⁶ differentways of combining the amino acids, and thus up to 20⁶ differentpeptides. The peptide library may also be a library comprising e.g. aspecific fraction of the amino acid sequence from a number of differentproteins with known sequences. The peptides must have lengths of atleast 6 amino acids in order to be able to bind to the hyper variableloops of the Fab fragment of the antibody. It follows that peptidelibraries may also be prepared using less than 20 amino acids—in whichcase of course the theoretical number of different combinations willvary accordingly.

Pharmaceutical carriers, excipients, or stabilizers which are non-toxicto the cell or mammal being exposed thereto at the dosages andconcentrations employed. Often the physiologically acceptable carrier isan aqueous pH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™

Plurality: At least two.

Polyclonal antibody: Polyclonal antibodies are a pool of immunoglobulinswhich arise from more than one clone of B-lymphocyte cells

Polypeptide derivatives: polypeptides derived from naturally occurringXXX by chemical modifications such as ubiquitination, labeling (e.g.,with radionuclides, various enzymes, etc.), pegylation (derivatizationwith polyethylene glycol), or by insertion (or substitution by chemicalsynthesis) of amino acids (amino acids) such as ornithine, which do notnormally occur in human proteins.

Pseudo-amino acid: an entity comprising a substituted amino group or/andcarboxyl group separated by an α-carbon or α-amine capable of beingincorporated into a peptide by ribosomes. For example, a pseudo aminoacid may comprise a thiol group and a carboxyl group separated by anα-carbon resulting in a thioester bond in the backbone.

Residue: A polymer comprises a sequence of covalently linked residues,wherein each residue comprises a functional group.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Surfactant: A surface active agents capable of reducing the surfacetension of a liquid in which it is dissolved. A surfactant is a compoundcontaining a polar group which is hydrophilic and a non polar groupwhich is hydrophobic and often composed of a fatty chain.

Vaccine/immunization cocktail/immunogenic composition: A substance orcomposition capable of inducing an immune response in an animal/host. Animmune response being an immune response (humoral/antibody and/orcellular) inducing memory in an organism, resulting in the generation ofantibodies against the introduced agents/antigens. The composition maycomprise one or more of the following: antigen(s), carriers, adjuvantsand pharmaceutical carriers.

Variant: a variant of a given reference nucleic acid or polypeptiderefers to a nucleic acid or polypeptide that displays a certain degreeof sequence homology to said reference nucleic acid or polypeptide butis not identical to said reference nucleic acid or polypeptide.

Vertebrate animal: A vertebrate animal is herein to be understood as ananimal from the vertebrate phylae. Preferred animals are animals fromeither the mammalian or the avian orders. Avian animals are particularlypreferred. Birds from the Galliformes order are most preferred. Birdsused for immunisation should have an age of at least 17 weeks,preferably at least 19 weeks.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an ELISA standard curve.

DETAILED DESCRIPTION OF THE INVENTION

The Method of Antibody Library Construction

The present invention relates to a novel method of constructing alibrary of antibodies in a fast and inexpensive manner by immunizingeach of a plurality of avian organisms of the same or a differentspecies with a composition comprising a plurality of different antigenscapable of raising in each avian organism a plurality of different IgYantibodies, and collecting said different IgY antibodies from eggs laidby said plurality of avian organisms.

Thus, in a first aspect, the present invention relates to a method ofobtaining polyclonal antibodies comprising antigen specific antibodies,wherein said method comprises the following steps:

(a) immunising a vertebrate animal with a composition comprising: (i)about 15 or more different peptide antigens, said peptides beingconjugated with a carrier and having a length of at least 6 amino acids,and (ii) an adjuvant,

(b) collecting a pool of polyclonal antibodies from the animal, whereina fraction of about 1% or more show reactivity towards the antigens usedfor immunisation.

The principle of immunising an animal, preferably a bird of thegallinacean type, and most preferably of the species Gallus gallusdomestica with a large amount of different antigens is a principle thatprovides tools for obtaining polyclonal antibodies in a much more fastand inexpensive way than previously known methods. The “antibodylibrary” can subsequently be used for isolating polyclonal antibodieswith a desired specificity. After having isolated specific polyclonalantibodies, the antibody library can be stored for later usage and thelibrary can thus be “recycled” in an (in theory) indefinite number oftimes. It follows, that it is much faster to isolate antigen specificantibodies from an antibody library which in theory comprises antibodiesthat have antibody binding capability to almost any antigen—rather thanimmunising the animal and waiting several weeks for a specific antibodyresponse.

A preferred way of obtaining an antibody library is by immunising with apeptide library obtainable by synthesizing peptides with a length of atleast 6 amino acids. Likewise, the peptide antigen may also be a mix ofpeptides with different lengths. The hen is ideal because it can produceabout 1500 mg antibodies per month (about 50-100 mg antibodies per egg(AOEA 24: 925-934, 1996). In fact, antibodies can be obtained in anon-invasive manner from the chicken during the entire egg-layingperiod. Usually, about 2-10% of the antibodies are antigen specific(AOEA 24: 925-934, 1996). The percentage of antigen specific antibodiesmay be even higher as a response to immunisation with a multitude ofantigens.

The antibody library thus constructed will in the first instance be alibrary of polyclonal antibodies comprising a plurality of antibodies ofdifferent specificities in accordance with the number of antigens thehost will be immunized with.

Prior to the construction of the antibody library, one or more librariesof antigens are synthesized. If the library is a peptide library thismay be done by random peptide synthesis. Subsequently the antigens maybe coupled to a carrier optionally in combination with one or moreadjuvants and used for the immunization of the host animals. The hostanimals are preferably the hens of an avian species. The antibodiesgenerated by each hen are isolated from the yolk of the eggs laid by theimmunized hens. All the antibodies thus collected are pooled and thispool constitutes a polyclonal antibody library. A monoclonal library mayalso be constructed as described in the below. From the antibodylibraries thus generated, antibodies specific for virtually any antigencan be isolated.

The antibody library can subsequently be used for isolating polyclonalantibodies with a desired specificity. The specificity does not have toreflect the antigens used during immunization. As the number ofantibodies of varying specificity is high, any compound may find itscomplementary antibody comprised within the library. In this manner,antibodies against compounds that otherwise may not or can not be usedfor immunization may be found. Specific antibodies can be harvested fromthe library according to the methods described in the below.Furthermore, the antibody library can be reused as a source of specificantibodies repeatedly, in theory an indefinite number of times. Itfollows, that it is much faster to isolate antigen specific antibodiesfrom an antibody library which in theory comprises antibodies that haveantibody binding capability to virtually any antigen—rather thanimmunizing the animal and waiting weeks to months for a specificantibody response. Thus the present invention provides a method ofobtaining specific polyclonal and/or monoclonal antibodies in a muchfaster and significantly less expensive way than is currently employed.

According to theory, a complete antibody library will containapproximately 5.8×10⁶ different antibodies as defined by differentbinding domains. This number is the result of the different possiblecombinations of the variable regions, heavy and light chains of theantibodies. From this number, the antibody binding domains thatrecognize species specific sequences must be subtracted, since a speciesnormally does not produce antibodies against itself. The exact numberthat has to be subtracted is unknown. It is expected that the immunesystem of an avian species reacts against at least 1.000 of 10.000introduced antigens. Similar reactions are observed when animals reactagainst different diseases at the same time. During immunization about 1in 1000 lymphoid cells are activated. The chicken immune system containsapp. 10⁹ lymphoid cells, and as one in a thousand in theory should beactivated, 1.000.000 cells are predicted to be able to react to amaximal stimulus. This is enough for reaction against 10.000 antigens.

The immunized birds are used for antibody production for an extendedperiod of time such as about one year during which egg-laying is at itsmaximum. After this period, the birds are killed and their bursas ofFabricius are harvested. This organ is a specialized organ that is thesite of hematopoiesis in birds, which is the residence of the antibodyproducing B cells. These antibody producing cells may be fused to amyeloma cell line, thus creating a hybridoma cell line that producesspecific antibodies. As each individual B cell and its immediatederivates—a specific B cell clone—only produce one type of antibody,these hybridoma cells are the source of monoclonal antibodies. In thismanner monoclonal antibodies can be harvested and pooled into a libraryof monoclonal antibodies.

By isolating specific antibody-producing cells employing thepurification method used for antibody purification it is possible toproduce monoclonal antibodies fast and cheap without difficult screeningprocedures and time consuming immunization. The production time isexpected to be around 14 days, and the costs of the monoclonalantibodies will be similarly reduced compared to traditional techniquesof isolating monoclonal antibodies.

A further advantage of using avian species for the rapid production ofantibody libraries, both poly- and monoclonal, is the avian specificimmunoglobulin molecule, IgY. Although the terms IgG and IgY arecommonly interchanged when speaking of chicken immunoglobulin, theaccepted term is IgY. Chicken IgY is the functional equivalent tomammalian IgG. Immunoglobulins from chickens and other avian speciesbear some resemblance to mammalian IgG, but also display some uniquestructural and functional characteristics that distinguish them fromIgG. Of similarities the following can be mentioned: IgY is divalent,degraded by papain to yield divalent Fab fragment and may be labeledi.e. enzyme-labeled, biotinylated and gold-labeled by standardprocedures. It is found in the serum of chickens and is passed from themother chicken to the embryo via the egg yolk, imparting a highconcentration of chicken IgY to developing embryo. The “Y” in IgY comesfrom “yolk” and is the main antibody component in the egg yolk. Eachyolk can yield 100-150 mg of antibody. As a measure of the amount ofantibodies that may be produced according to the present invention,consider the following: A chicken produces around 100 mg IgY/egg, whichcorresponds to 30 g IgY per year. Depending on the number of birdsimmunized, kilograms of polyclonal antibodies may thus be harvested.Furthermore, the IgY molecule is more stable than the traditionally usedIgG molecules and IgY has less cross-reactivity toward mammalianproteins. IgY does not bind rheumatoid factors or Fc-receptors norproteins A or G, therefore the chicken's antibodies are unlikely toproduce false positive reactions in certain immunochemical assays. Also,unlike mice, rabbits or other mammals, hens elicit a strong antibodyresponse against highly conserved mammalian protein sequences, and theIgY's can be obtained without sacrificing or bleeding the animal.

Antigens

The antigens of the present invention are may comprise any compoundcapable of eliciting an immune response in an animal, said animalpreferably being an avian. Examples of compounds thus included arenatural and non-natural amino aid residues, and combinations of these asnatural amino acids only, as non-natural amino acids only or combinationof either together into peptides or peptide-like compounds. Among thenon-natural amino acids are the “unnatural” amino acids described byMendel et al., (Annu. Rev. Biophys. Biomol. Struct. 1995 24:435-62—thedocument is hereby incorporated by reference and of special relevancefor the present invention are the amino acids illustrated in FIG. 3 ofsaid document). Be noted, that among these non-natural amino acids areacids, that do not comprise an amino (—NH2) group, these are alsoincluded as moieties of the present invention. Preferably, the moietiesof the present invention are naturally occurring amino acids.

The naturally occurring amino acids of the invention may besynthetically prepared and linked or may be the result of proteolyticcleavage of any type of protein that may be isolated from any type oforganism. An aspect of the present invention relates to antigens and/orproteins derived from organisms. These organisms may be any organism,such as pathogenic or non-pathogenic organisms. Especially if pathogenicorganisms are used as a source of antigens, they may be inactivated orkilled prior to use by heat inactivation, autoclaving or any other meansknown to the art. Naturally occurring peptides may furthermore bemutated by the substitution, addition or deletion of natural ornon-natural amino acid residues. Especially substitutions may be benign,meaning that an amino acid residue is exchanged for an amino acidresidue of the same type such as the original. As an example: The aminoacids Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys all havepolar chains and thus exchanging one of these for another wouldconstitute a benign substitution. Examples of various groups of aminoacids that are related by the nature of their side chains apart from theabovementioned are: Amino acids having non-polar side chains (Gly, Ala,Val, Leu, Ile, Phe, Trp, Pro, and Met), amino acids having aliphaticside chains (Gly, Ala Val, Leu, Ile), amino acids having cyclic sidechains (Phe, Tyr, Trp, His, Pro), amino acids having aromatic sidechains (Phe, Tyr, Trp), amino acids having acidic side chains (Asp,Glu), amino acids having basic side chains (Lys, Arg, His), amino acidshaving amide side chains (Asn, Gln), amino acids having hydroxy sidechains (Ser, Thr), amino acids having sulphor-containing side chains(Cys, Met), neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,Thr), hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), andhydrophobic amino acids (Leu, Ile, Val). Substitutions may also occuroutside of any of the above defined groups. The resulting peptide may bereferred to as a mutated peptide.

The moieties of the present invention may furthermore be modified inseveral ways. These modifications are often referred to as posttranslational modifications, as the modifications take place after thetranslation of the mRNA into protein within a cell. All of the mentionedmodifications may be performed synthetically on any of the moieties ofthe invention. The modifications of the present invention include butare not limited to modifications involving the addition of functionalgroup such as: acetylation (the addition of an acetyl group, usually atthe N-terminus of the protein), alkylation (the addition of an alkylgroup (e.g. methyl, ethyl), especially methylation (the addition of amethyl group, usually at lysine or arginine residues), biotinylation(acylation of conserved lysine residues with a biotin appendage),glutamylation (covalent linkage of glutamic acid residues to tubulin andsome other proteins), glycylation (covalent linkage of one to more than40 glycine residues to the tubulin C-terminal tail), glycosylation (theaddition of a glycosyl group typically to either asparagine,hydroxylysine, serine, or threonine, resulting in a glycoprotein)isoprenylation (the addition of an isoprenoid group e.g. farnesol andgeranylgeraniol), lipoylation (attachment of a lipoate functionality),phosphopantetheinylation (the addition of a 4′-phosphopantetheinylmoiety from coenzyme A, as in fatty acid, polyketide, non-ribosomalpeptide and leucine biosynthesis), phosphorylation (the addition of aphosphate group to any residue, but typically to serine, tyrosine,threonine or histidine), sulfation (the addition of a sulfate group to atyrosine), selenation and C-terminal amidation.

Preferably, the moieties of the present invention are modified byphosphorylation, sulfation, glycosylation, methylation, and/oracetylation. Any moiety may thus be modified at least once with any ofthe above mentioned modifications. And any peptide or peptide-likecompound may comprise none, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20 ormore modifications or any number herein between or above.

Other modifications may be the addition of other proteins or peptidessuch as ISGylation (the covalent linkage to the ISG15 protein(Interferon-Stimulated Gene 15)), SUMOylation (the covalent linkage tothe SUMO protein (Small Ubiquitin-related MOdifier)), ubiquitination(the covalent linkage to the protein ubiquitin), or may involve a changeof the chemical nature of amino acids, such ascitrullination/deimination (the conversion of arginine to citrulline),deamidation (the conversion of glutamine to glutamic acid or asparagineto aspartic acid) or modifications may involve structural changes suchas disulfide bridges (the covalent linkage of two cysteine amino acids),proteolytic cleavage (cleavage of a protein at a peptide bond).Preferably, the moieties of the present invention are modified byubiquitination, deamidation or the formation of disulfide bridges. Thesemodifications may be singularly appearing or repeatedly appearingtogether with any of the above mentioned modifications.

By peptide is meant any plurality of individual moieties as arecomprised in the above definition, occasionally these will be referredto as peptide-like compounds in the present text. Thus any type of bondmay link the individual moieties of above, not only peptide bonds.Examples of bonds linking moieties of the present invention are: peptidebonds, sulfonamide bonds, ester bonds, saccharide bonds, carbamatebonds, carbonate bonds, urea bonds, phosphonate bonds, urethane bonds,azatide bonds, peptoid bonds, ether bonds, ethoxy bonds, thioetherbonds, single carbon bonds, double carbon bonds, triple carbon bonds,disulfide bonds, sulfide bonds, phosphodiester bonds, oxime bonds, iminebonds and imide bonds. Any of the bonds of the above may be used as theonly type of bond within the peptide, or two or more such as three ormore different types of bonds may be comprised within a peptide-likecompound. Preferably, the bonds connecting the moieties of the inventionare peptide bonds, imine bonds and imide bonds, and most preferablypeptide bonds.

The peptides of the present invention may furthermore comprise variousdomains such as an α-peptide domain, a β-peptide domain, a γ-peptidedomain, a ω-peptide domain, a mono-, di- and tri-substituted α-peptidedomain, a mono-, di- and tri-substituted β-peptide domain, a mono-, di-and tri-substituted γ-peptide domain, a mono-, di- and tri-substitutedω-peptide domain, a peptide domain wherein the amino acid residues arein the L-form, a peptide domain wherein the amino acid residues are inthe D-form, a peptide domain wherein the amino acid residues are both inthe L-form and in the D-form, a vinylogous peptide domain, aglyco-peptide domain, a vinylogous sulfonamide peptide domain, apolysulfonamide domain, a conjugated peptide domain and a conjugatedpeptide domain comprising one or more functional group(s), such as oneor more prosthetic group(s).

Antigens according to the present invention are defined as antigens witha length in the range of from 5 moieties as defined in the above topreferably less than 1000 moieties. The length of the antigens may thusbe at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20moieties or amino acid residues long, or at least 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,600, 650, 700, 750, 800, 850, 900, 950 or 1000 moieties in length.Preferably, the antigens are between 5 and 500 moieties in length or anyinterval comprised therein, such as between, 5 and 400, 5 and 300, 5 and200, 5 and 100, 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and25, 5 and 24, 5 and 23, 5 and 22, 5 and 21, 5 and 20, 5 and 19, 5 and18, 5 and 17, 5 and 16, 5 and 15, 5 and 14, 5 and 13, 5 and 12, 5 and11, 5 and 10, 5 and 9, 5 and 8, 5 and 7 or 6 moieties in length. Theantigens may furthermore be of a length in the range of from 10 to 20,10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10to 100, 10 to 125, 10 to 150, 10 to 175, 10 to 200 moieties in length orfrom 15 to 20, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 15 to 60, 15 to70, 15 to 80, 15 to 90, 15 to 100, 15 to 125, 15 to 150, 15 to 175, 15to 200, 20 to 50, 20 to 75, 20 to 100, 20 to 200, 20 to 250, 20 to 300,20 to 350, 20 to 400 or more in length.

The antigens of the present invention are typically comprised in alibrary of antigens, meaning that there are more than 2 antigenspresent. The antigens according to the invention can be a mix ofantigens and/or peptides of different lengths, i.e. there may be shortantigens and/or peptides present with a length as short as 5 moieties aswell as longer antigens. Some or all of the antigens/peptides may alsobe in form of peptides/polypeptides or proteins being either denaturedor in their naturally folded form. It follows that proteins can beobtained from either a natural source or they can be recombinantlyproduced and subsequently purified.

Antigens according to the present invention are preferably of peptideorigin with a length of at least 5 amino acids, and may be derivedeither from natural sources, produced by recombinant methods, orsynthesized artificially—or any mixtures thereof. The antigens used forimmunisation in connection with the present invention may comprise or besubstantially free from e.g. contaminating cellular debris such ascellular material including cell membranes, nucleic acids, etc.

Carriers

Conjugation to a carrier is important because antigens, peptides andpeptide-like compounds may be small molecules and small molecules alonedo not tend to be immunogenic, thus possibly eliciting a weak immuneresponse. The carrier typically contains many epitopes that stimulateT-helper cells, which help induce the B-cell response.

The carrier may be present independently of an adjuvant (please see inthe below). The purpose of conjugation and/or co-immunization of anantigen/immunogenic determinant and a carrier can be e.g. to increasethe molecular weight of the antigen in order to increase the activity orimmunogenicity of the determinant, to confer stability to thedeterminant, to increase the biological activity of the determinant, orto increase its serum half-life. The carrier protein may be anyconventional carrier including any protein suitable for presentingimmunogenic determinants. Conventional carrier proteins include, but arenot limited to, keyhole limpet hemocyanin, serum proteins such astransferrin, bovine serum albumin, or human serum albumin, an ovalbumin,immunoglobulins, or hormones, such as insulin.

While any suitable pharmaceutical carrier known to those of ordinaryskill in the art may be employed in the immunogenic and pharmaceuticalcompositions of this invention, the type of pharmaceutical carrier willvary depending on the mode of administration and whether a sustainedrelease administration is desired. For parenteral administration, suchas subcutaneous injection, the pharmaceutical carrier may e.g. comprisewater, saline, alcohol, fat, a wax or a buffer. For oral administration,any of the above pharmaceutical carriers or a solid pharmaceuticalcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as pharmaceutical carriers for thepharmaceutical compositions of this invention. Suitable biodegradablemicrospheres are disclosed, for example, in U.S. Pat. No. 4,897,268 andU.S. Pat. No. 5,075,109.

The antigen may be encapsulated within the biodegradable microsphere orassociated with the surface of the microsphere.

Antigens within the scope of the present invention could be conjugatedby any method known to the person skilled in the art. For example theycould be conjugated by a physical association generated for example bythe formation of a chemical bond, such as for example a covalent bond,formed between compounds to be conjugated. Compounds could be conjugatedfor example by an oxidative induced cross-link, such as mild oxidativeinduced cross-link catalyzed by long-time expo-sure to atmospheric air,such as an over-night exposure. Alternatively, compounds could beconjugated using a chemical cross-linking reagent. Examples of chemicalcross-linking reagents are glutaraldehyde, charbodiimid or formaldehyde.

The present invention provides embodiments comprising one or moreantigens according to the above which are non-conjugated, an immunogeniccomposition wherein said antigen is conjugated and an immunogeniccomposition wherein said antigens are a mixture of non-conjugated andconjugated antigens.

Many different carriers can be used for coupling to antigens, especiallyto synthetic peptides. The most commonly selected carriers here fore arekeyhole limpet hemacyanin (KLH) and bovine serum albumin (BSA). Thehigher immunogenicity of KLH often makes it the preferred choice.Another advantage of choosing KLH over BSA is that BSA is used as ablocking agent in many experimental assays. Because antisera raisedagainst peptides conjugated to BSA will also contain antibodies to BSA,false positives may result. Although KLH is large and immunogenic, itmay precipitate during cross-linking, making it difficult to handle insome cases. Ovalbumin (OVA) is another useful carrier protein. It is agood choice as a second carrier protein when verifying whetherantibodies are specific for the peptide alone and not the carrier.Rabbit Serum Albumin (RSA) may be used when the antibody response to thecarrier protein must be kept to a minimum. Rabbits immunised with RSAconjugate are less likely to raise antibodies to the carrier, as the RSAis recognized as “self.” If the RSA conjugate were injected into anotherhost, the protein would not be recognized as self. The same principle,of course, applies when immunising with chicken-derived carriers inchicken. An example of a chicken derived carrier is CSA (Chicken SerumAlbumin). By using CSA (Chicken Serum Albumin) or beads an immuneresponse against the carrier may be avoided, thereby optimizing theproductivity of the chicken immune response. It is important torecognize that the immune system reacts to the peptide-protein carrieras a whole and that there will be a portion of response directed againstthe conjugated peptide as well as the linker and the carrier. Whenscreening by ELISA it is advisable to use a peptide conjugate preparedusing a different carrier. This is not necessary if performing ELISAassays where the plates are coated directly with unconjugated peptide.Thus examples of carriers include, but are not limited to: BSA, CSA, SA,KLH, Beads or other carrier-systems. Suitable carriers will be ofprotein origin in most cases. In some cases however, the carrier may beof non-protein origin such as e.g. polymers comprising carbohydrates,etc. Preferably either CSA or beads are used to avoid immune responseagainst the carrier.

It is an aspect of the present invention to use the carrier KLH.

The coupling can be made by glutaraldehyde, MBS or other couplingmethods. Glutaraldehyde couples between free amino groups (—NH2) andcysteine. The ratio between peptide and carrier has to be exact to avoidover-coupling. By decreasing the pH to a value lower than the pK valueof the amino acids coupling to the side chains is avoided. Correctionhas to be made for sequences containing cysteine.

It may not be necessary to conjugate relatively large antigens peptideswith a carrier in order to obtain an immune response. Antigens/peptideswith a length of at least 50 moieties/amino acids and more, preferablywith a length of at least 100 moieties or at least 150 moieties may notneed carrier conjugation in order to function as immunogens in thepresent invention. It is however advisable to conjugate all antigenswith carrier molecules disregard the size of the antigen/the antigens.

Antigen Library

It is an object of the present invention that the plurality of avianspecies each is immunized with a plurality of antigens, herein referredto as an antigen library. The antigens of the library may or may not becoupled to a carrier as described in the above. The average numbers ofantigens within such a library is in the range of from 2 to 100000.

In another aspect of the present invention polyclonal antibodiescomprising antigen specific antibodies are obtained, by immunising avertebrate animal with a composition comprising about 15 or moredifferent peptide antigens, said peptides being conjugated with acarrier and having a length of at least 6 amino acids, and an adjuvant,collecting polyclonal antibodies from the animal, wherein a fraction ofabout 1% or more shows reactivity towards the antigens used forimmunisation.

It is an aspect of the present invention to use an animal that isreimmunised.

In an embodiment of the present invention antibodies are collected fromsaid animal after a period of at least 14 days.

In one aspect of the invention the peptide antigens is a peptide libraryobtainable by randomly synthesizing peptides with a length of at least 6amino acids.

It is an aspect of the present invention to use an antigen mixcomprising peptides with different lengths.

An antigen library may be a peptide library constructed randomly bysolid-phase-synthesis with controlled coupling to eithertert-butyl-oxy-carbonyl (BOC) groups or Fluorenyl-metoxy-carbonyl(FMOC). Instead of producing the single peptides individually, they areall produced simultaneously. Peptides will be produced to cover allpossible domains. This means that modified moieties such as glycosylatedand phosphorylated moieties/amino acids also are represented. Employingthe random coupling between the amino acids the peptides are producedfast, cheap and divided into smaller groups. A smaller group refers asan example to 10.000 antigens. A group can be generated by excludingcertain moieties or modifications from the reaction.

A way of carrying out the present invention is to synthesize a peptidelibrary with peptides of similar lengths covering all the possiblecombinations of the moieties disclosed in the above. Peptide synthesismay take place in solution or on a solid phase. A peptide library withpeptides of a length of e.g. 6 of the 20 most often naturally occurringamino acids will thus in theory have 20⁶ different ways of combining theamino acids, and thus up to 20⁶ different peptides. The peptide librarymay also be a library comprising e.g. a specific fraction of the aminoacid sequence from a number of different proteins with known sequences.The peptides must have lengths of at least 5 amino acids in order to beable to bind to the hypervariable loops of the Fab fragment of theantibody.

It follows that peptide libraries may also be prepared using more orless than the 20 most often occurring natural amino acids—in which caseof course the theoretical number of different combinations will varyaccordingly. Any of the above defined antigens comprising any of theabove defined moieties with any of the above defined modifications maybe combined to form part of the antigen library. Any of the resultingcombinations may or may not be coupled to a carrier according to theabove prior to immunization (with or without further mixing with anadjuvant).

Host

The host is the animal or individual used for the generation of theantibody libraries according to the present invention. The host may bean animal from the vertebrate phyla. Preferred animals are animals fromeither the mammalian or the avian orders. Avian animals are particularlypreferred. Birds from the Galliformes order are most preferred, thus inone embodiment of the present invention the vertebrate animal is of thespecies Gallus gallus domestica.

The Galliformes order of birds includes grouse, ptarmigan, capercaillie,partridges, pheasants, quails, turkeys and peacocks. These are mainlygrain-eating, heavy-bodied, ground-nesting birds, capable of only short,rapid flights. The cocks are usually more colourful than the hens. Apreferred bird according to the present invention is a Gallus gallusdomestica either in the adult stage or as a chicken at the age of atleast 17-19 weeks. Especially preferred are the females of any of theabove mentioned avian species. Birds used for immunisation should havean age of at least 17 weeks, preferably at least 19 weeks.

The hen of Gallus gallus domestica is ideal because it can produce about1500 mg antibodies per month (about 50-100 mg antibodies per egg (AOEA24: 925-934, 1996). In fact, antibodies can be obtained in anon-invasive manner from the chicken during the entire egg-layingperiod. Usually, about 2-10% of the antibodies are antigen specific(AOEA 24: 925-934, 1996). The percentage of antigen specific antibodiesmay be even higher as a response to immunization with a multitude ofantigens.

It is an important aspect of the present invention to immunize aplurality of hosts for the production of antibody libraries. Therefore,the number of host, especially avian organisms, immunized is about 1000,such as about 2000, for example about 3000, such as about 4000, forexample about 5000, such as about 5500, for example about 6000, such asabout 6500, for example about 7000, such as about 75000, for exampleabout 8000, such as about 8500, for example about 9000, such as about9500, for example about 10000, such as about 10500, for example about11000, such as about 115000, for example about 12000, such as about12500, for example about 13000, such as about 13500, for example about14000, such as about 14500, for example about 15000, such as about16000, for example about 17000, such as about 18000, for example about19000, such as about 20000, for example about 21000, such as about22000, for example about 23000, such as about 24000, for example about25000, such as about 26000, for example about 27000, such as about28000, for example about 29000, such as about 30000, for example about31000, such as about 32000, for example about 33000, such as about34000, for example about 35000, such as about 36000, for example about37000, such as about 38000, for example about 39000, such as about40000, for example about 41000, such as about 42000, for example about43000, such as about 44000, for example about 45000, such as about50000.

Preferably, any number of hosts from 10 to 100000 is immunized. Morepreferably the number of hosts is between 100 and 50000, such as between200 and 40000, 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000,800 and 6000, 900 and 5500, 1000 and 5000.

Adjuvants

Adjuvants, also known as pharmaceutical carriers, or functionalequivalents hereof may be included in the immunization solution/vaccinecomposition to enhance the specific immune response. Thus, it isparticular important to identify an adjuvant that when combined with theantigen(s) results in an immunization composition capable of inducing astrong specific immunological response. Functionally equivalent carriersare capable of presenting the same immunogenic determinant inessentially the same steric conformation when used under similarconditions. Functionally equivalent adjuvants are capable of providingsimilar increases in the efficacy of the composition when used undersimilar conditions.

Preferably, the immunogenic compositions (composition of the presentinvention) comprise potent, nontoxic adjuvants that will enhance and/ormodulate the immunogenicity of immunogenic determinants includingantigenic determinants including haptenic determinants represent onegroup of preferred adjuvants. In addition, such adjuvants preferablyalso elicit an earlier, more potent, or more prolonged immune response.Such an adjuvant would also be useful in cases where an antigen supplyis limited or is costly to produce.

A large number of adjuvants have been described and used for thegeneration of antibodies in laboratory animals, such as mouse, rats,rabbits and chickens. In such setting the tolerance of side effect israther high as the main aim is to obtain a strong antibody response.

An embodiment of the present invention relates to an immunizationcomposition comprising an adjuvant. In a preferred embodiment thevaccine composition is suitable for administration to an avian subject,thus a preferred adjuvant is suitable for administration to a chicken orhen as defined in the above.

The choice of adjuvant may further be selected by its ability tostimulate the type of immune response desired, B-cell or/and T-cellactivation and the vaccine composition may be formulated to optimizedistribution and presentation to the relevant lymphatic tissues.

Adjuvants pertaining to the present invention may be grouped accordingto their origin, be it mineral, bacterial, plant, synthetic, or hostproduct. The first group under this classification is the mineraladjuvants, such as aluminum compounds. Antigens precipitated withaluminum salts or antigens mixed with or adsorbed to performed aluminumcompounds have been used extensively to augment immune responses inanimals and humans. Aluminium particles have been demonstrated inregional lymph nodes of rabbits seven days following immunization, andit may be that another significant function is to direct antigen to Tcell containing areas in the nodes themselves. Adjuvant potency has beenshown to correlate with intimation of the draining lymph nodes. Whilemany studies have confirmed that antigens administered with aluminiumsalts lead to increased humoral immunity, cell mediated immunity appearsto be only slightly increased, as measured by delayed-typehypersensitivity. Aluminium hydroxide has also been described asactivating the complement pathway. This mechanism may play a role in thelocal inflammatory response as well as immunoglobulin production and Bcell memory. Furthermore, aluminum hydroxide can protect the antigenfrom rapid catabolism. Primarily because of their excellent record ofsafety, aluminum compounds are presently the only adjuvants used inhumans.

Another large group of adjuvants is those of bacterial origin. Adjuvantswith bacterial origins can be purified and synthesized (e.g. muramyldipeptides, lipid A) and host mediators have been cloned (Interleukin 1and 2). The last decade has brought significant progress in the chemicalpurification of several adjuvants of active components of bacterialorigin: Bordetella pertussis, Mycobacterium tuberculosis,lipopoly-saccharide, Freund's Complete Adjuvant (FCA) and Freund'sIncomplete Adjuvant (Difco Laboratories, Detroit, Mich.) and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Additionallysuitable adjuvants in accordance with the present invention are e.g.Titermax Classical adjuvant (SIGMA-ALDRICH), ISCOMS, Quil A, ALUN, seeU.S. Pat. Nos. 58,767 and 5,554,372, Lipid A derivatives, choleratoxinderivatives, HSP derivatives, LPS derivatives, synthetic peptidematrixes, GMDP, and other as well as combined with immunostimulants(U.S. Pat. No. 5,876,735).

B. pertussis is of interest as an adjuvant in the context of the presentinvention due to its ability to modulate cell-mediated immunity throughaction on T-lymphocyte populations. For lipopolysaccharide and Freund'sComplete Adjuvant, adjuvant active moieties have been identified andsynthesized which permit study of structure-function relationships.These are also considered for inclusion in immunogenic compositionsaccording to the present invention.

Lipopolysaccharide and its various derivatives, including lipid A, havebeen found to be powerful adjuvants in combination with liposomes orother lipid emulsions. It is not yet certain whether derivatives withsufficiently low toxicity for general use in humans can be produced.Freund's Complete Adjuvant is the standard in most experimental studies.

Mineral oil may be added to the immunogenic composition in order toprotect the antigen from rapid catabolism.

Many other types of materials can be used as adjuvants in immunogeniccompositions according to the present invention. They include plantproducts such as saponin, animal products such as chitin and numeroussynthetic chemicals.

Adjuvants according to the present invention can also been categorizedby their proposed mechanisms of action. This type of classification isnecessarily somewhat arbitrary because most adjuvants appear to functionby more than one mechanism. Adjuvants may act through antigenlocalization and delivery, or by direct effects on cells making up theimmune system, such as macrophages and lymphocytes. Another mechanism bywhich adjuvants according to the invention enhance the immune responseis by creation of an antigen depot. This appears to contribute to theadjuvant activity of aluminum compounds, oil emulsions, liposomes, andsynthetic polymers. The adjuvant activity of lipopolysaccharides andmuramyl dipeptides appears to be mainly mediated through activation ofthe macrophage, whereas B. pertussis affects both macrophages andlymphocytes. Further examples of adjuvants that may be useful whenincorporated into immunogenic compositions according to the presentinvention are described in U.S. Pat. No. 5,554,372.

Useful adjuvants useful may thus be mineral salts, such as aluminiumhydroxide and aluminium or calcium phosphates gels, oil emulsions andsurfactant based formulations such as MF59 (microfluidised detergentstabilised oil in water emulsion), QS21 (purified saponin), AS02 (SBAS2,oil-in-water emulsion+monophosphoryl lipid A (MPL)+QS21), Montanide ISA51 and ISA-720 (stabilised water in oil emulsion), Adjuvant 65(containing peanut oil, mannide monooleate and aluminum monostearate),RIBI ImmunoChem Research Inc., Hamilton, Utah), particulate adjuvants,such as virosomes (unilamellar liposomal cehicles incorporatinginfluenza haemagglutinin), AS04 (Al salt with MPL), ISCOMS (structuredcomplex of saponins and lipids (such as cholesterol), polyactideco-glycolide (PLG), microbial derivatives (natural and synthetic) suchas monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wallskeleton), AGP (RC-529 (synthetic acylated monosaccharide)), DC_chol(lipoidal immunostimulators able to self organise into liposomes),OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotidescontaining immunostimulatory CpG motifs), modified bacterial toxins, LTand CT, with non-toxic adjuvant effects, Endogenous humanimmunomodulators, e.g., hGM-CSF or hIL-12 or Immudaptin (C3d tandemarray), inert vehicles such as gold particles. Additional examples ofadjuvants comprise: Immunostimulatory oil emulsions (for example,water-in-oil, oil-in-water, water-in-oil-in-water such as e.g. Freund'sincomplete adjuvant such as Montainde®, Specol, mineral salts such e.g.as Al(OH)₃, AIPO₄, microbial products, Saponins such as Qual A,synthetic products, as well as adjuvant formulations, and immunestimulatory complexes (ISCOMs) and cytokines, heat-inactivatedbacteria/components, nanobeads, LPS, LTA. A list of other commonly usedadjuvants is disclosed on pages 6-8 in WO 03089471, the list beinghereby incorporated by reference.

Immunogenic compositions according to the invention may also containdiluents such as buffers, antioxidants such as ascorbic acid, lowmolecular weight (less than about 10 residues) polypeptides, proteins,amino acids, carbohydrates including glucose, sucrose or dextrins,chelating agents such as EDTA, glutathione and other stabilizers andexcipients. Neutral buffered saline or saline mixed with non-specificserum albumin are exemplary appropriate diluents. Preferably, thecompostions are formulated as a lyophilizate using appropriate excipientsolutions (e.g., sucrose) as diluents.

The immunogenic compositions of the present invention may furthermorecomprise pharmaceutical carriers. Suitable carriers are typically large,slowly metabolized macromolecules such as proteins, polysaccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Such carriers are well known to those ofordinary skill in the art. Additionally, these carriers may function asimmunostimulating agents (“adjuvants”). Immunisation of the animal maybe carried out with adjuvants and/or pharmaceutical carriers. Anadjuvant is any substance that enhances the immune response to anantigen with which it is mixed. The antigen may also be mixed with twoor more different adjuvants prior to immunisation.

It is an aspect of the present invention to utilize the adjuvant Specol.

Adjuvants are generally included in the immunogenic compositions in anamount according to the instructions of the manufacturer. If theadjuvant is Specol an amount of e.g. 1:1 compared to the antigen/carriermixture or antigen mixture alone may be added to the immunogeniccomposition.

Preferably, the adjuvants of the present invention are Freunds completeadjuvant, Freunds incomplete adjuvant, Specol, Titermax Gold,Alhydrogel, Gerbu Adjuvant 100, Diluvac Forte and Super Adjuvant asdefined in the Examples. More preferably, the adjuvants are Freundscomplete adjuvant, Freunds incomplete adjuvant, Specol, Titermax Gold,and Super Adjuvant. With these preferred adjuvants, antibodies may beharvested after only about two weeks, instead after three to six weeks.

Method of Immunization

Immunogenic compositions according to the invention may be administeredto a host in effective amounts resulting in the optimum production ofantibodies. The effective amount may vary according to a variety offactors such as the host's condition, weight, and age. Other factorsinclude the mode of administration.

The immunogenic compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tissue), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. The present inventionalso has the objective of providing suitable topical, oral, systemic andparenteral pharmaceutical formulations for use in the methods ofprophylaxis and treatment of the vaccine composition.

For example, the immunogenic compositions can be administered in suchoral dosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the vaccine, comprising any of the herein describedcompounds can be employed as a prophylactic or therapeutic agent. Alsoany and all conventional dosage forms that are known in the art to beappropriate for formulating injectable immunogenic peptide compositionare encompassed, such as lyophilised forms and solutions, suspensions oremulsion forms containing, if required, conventional pharmaceuticallyacceptable carriers, diluents, preservatives, adjuvants, buffercomponents, etc.

Preferred modes of administration of the immunogenic compositionaccording to the invention include, but are not limited to systemicadministration, such as intravenous or subcutaneous administration,intradermal administration, intramuscular administration, intranasaladministration, oral administration, rectal administration, vaginaladministration, pulmonary administration and generally any form ofmucosal administration. Furthermore, it is within the scope of thepresent invention that the means for any of the administration formsmentioned in the herein are included in the present invention.

Preferably, the method of administration involves injection. The animalsmay be immunised with the antigens intramuscularly, intrasplenically,intravenously, intraperitoneally, intradermally or subcutaneously or byany other suitable means. Most preferably the chickens kept under fieldconditions are immunized in the breast musculature with a pistol. Thisspares the chicken for a slow and stressing immunization. In thelaboratory, chickens most preferably are vaccinated subcutaneously inthe neck.

An immunogenic composition according to the present invention can beadministered once, or any number of times such as two, three, four orfive times. Administering the composition more than once has the effectof boosting the resulting immune response. The composition can furtherbe boosted by administering it in a form or body part different from theprevious administration. The booster shot is either a homologous or aheterologous booster shot. A homologous booster shot is a where thefirst and subsequent administrations comprise the same compositions andmore specifically the same antigens and adjuvants. A heterologousbooster shot is where identical antigen libraries are comprised indifferent compositions, i.e. different adjuvants and carriers.

The total number of immunisations will depend upon the type and dose ofthe antigen, as well as on the particular adjuvant and carrier employed.In a preferred embodiment, at least two administrations are given, suchas 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. If the antibodytitres begin to decrease, further booster immunisations can be given.

It falls within the scope of the present invention that the means andmodes of administration of the immunogenic composition are adapted tothe host. A preferred recipient of the vaccine is an avian, such as achicken of the species Gallus gallus.

An embodiment of the present invention includes an immunogeniccomposition further comprising a second active ingredient. The secondactive ingredient is selected from, but not limited the group ofantibiotics, chemotherapeutics, anti-allergenics, cytokines, complementfactors and co-stimulatory molecules of the immune system, any compoundor supplement which may be of benefit to the host organism.

The total amount of peptide employed for each immunisation may likewisevary. Amounts of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 μg arepreferably used in a single immunisation. But amounts of about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250,or 500 mg may also be used in a single immunisation.

The method of any of claims 1 to 25 further comprising administeringsaid different antigens to said avian organism(s) in the presence of oneor more carrier(s) covalently or non-covalently attached to at leastsome of said antigens.

The amount of different peptide antigens used for immunisation of theanimal is at least 15. As the aim of the present invention is to providetools for rapidly isolating antigen specific antibodies, it isadvantageous to immunise with even more antigens, as the likelihood thusincreases of obtaining antibodies with specificity to most antigens. Itis therefore preferable to immunise with at least about 25, 50, 51, 60,70, 80, 90, 100, 150, 200, 250, 500, 1000, 5000, 10000, 50000, 100000,500000, 1000000, or 5000000 different antigens. It is most preferable toimmunise with a synthetically synthesized peptide library comprising, intheory, 20⁶ or more different peptide combinations.

Antibody Library

The present invention relates to a method for generating an antibodylibrary i.e. a plurality of different IgY antibodies, said methodcomprising the step of immunizing each of a plurality of avian organismsof the same or different species with a composition comprising aplurality of different antigens, thereby generating a plurality ofdifferent IgY antibodies

A preferred way of obtaining an antibody library is by immunizing with apeptide library obtainable by synthesizing peptides with a length of atleast 5 amino acids. Likewise, the peptide antigen may also be a mix ofpeptides with different lengths.

A library of antibodies according to the present invention can beobtained by immunising a single animal with an antigen library, forexample a randomly generated peptide library. A randomly generatedpeptide library will in general stimulate an antibody responsecomprising antibodies with binding capability of (in theory) anyantigen.

According to the present invention an antibody can be termed antigenspecific if e.g. antigen:antibody binding can be demonstrated in anassay as disclosed in the examples, where antigen is linked to a carrierin the presence of PBS and antibodies are likewise suspended in a PBSbuffer.

According to the present invention, at least about 1% of the antibodiesobtained subsequent to immunisation show specificity to the antigensused for immunisation. If less than 1% of the antibodies showspecificity to the antigens, antibodies may e.g. have been harvested toosoon after immunisation, insufficient amounts of antigen have beenemployed, or inefficient amounts of adjuvants have been employed. Morepreferably, at least about 2, 3, 4, or 5% of the antibodies are antigensspecific. Most preferably, at least about 6, 7, 8, 9, or 10% of theantibodies are specific. And most preferably about 20-30% of theantibodies are antigen specific.

Conditions suitable for an antigen:antibody binding to take place: suchconditions are well known in the art. In the Examples, PBS buffer isemployed.

It is known in the art how to purify antibodies from egg yolk. Pleasesee the examples for methods hereof. Of additional methods hereofinclude the use of Dextran, Lithium sulfat, Triton, CA, Xan asprecipitation agents or the use of the method of water dilution. Allprecipitated and thus harvested antibodies are pooled into the antibodylibrary.

Harvest of Antibodies

The antigen that one wishes to have a specific antibody against (i.e.with binding specificity towards) is synthesized an coupled/linked to anepoxy coated pole. The pole is the transferred into the antibody librarystorage device, wherein an interaction between the antigen(s) andantibodies will take place. The pole is removed from the library and byvarying the strength (lowering the pH) of the elution solution or bysonication at varying strengths, the bound antibodies can be elutedaccording to their binding affinity, which may be termed: low medium andhigh. The antigen is covalently bound to the pole, and the bound to theantibody by ion-bonds (hydrogen bonds aso.), wherefore the bond betweenthe antigen and antibody is broken first. The fluid comprising theantibodies is dialyzed against PBS, and any reduction in the volume iscorrected. The desired antibody, it being of high, medium or lowaffinity according to ones needs, is now available as an isolatedproduct. Any unwanted antibodies may be returned to the library.

In an embodiment of the present invention antigen specific antibodiesare isolated from an antibody pool generated as described herein above,wherein the method comprises the steps of immobilising the antigen,contacting the antibody pool with the immobilized antigen underconditions suitable for an antigen:antibody binding to take place,removing unbound antibody, and recovering antigen specific antibodies.

In another embodiment of the present invention the antigen specificantibodies described herein above are recovered using acidic conditions.

It is an object of the present invention to immobilize the antigen on aCNBr carrier, such that the peptide or antigen that one wishes to have aspecific antibody against is synthesised and coupled to sepharose-CNBrbeads. The beads are then transferred to a column and the antibodylibrary or a fraction hereof will be run over the column. Antibodieswill bind to the antigens bound to the column matrix. By eluting theantibodies with decreasing values of pH, antibodies of first low,subsequently medium and finally high affinity will be eluted from thecolumn.

The antibody:antigen bound beads may subsequently (after or beforeelution with varying pH strengths) by transferred to a flow chamber. Inthe flow chamber sound pulses (sonication) will release the most tightlybound antibodies from the antigens. The flowthrough comprising the thusreleased antibodies is dried and dialysed against PBS.

Any superfluous antigen/peptide is stored for refilling the library.Refilling the library may be by immunizing a number of hosts with theparticular antigen/peptide and adding the thus generated antibodies tothe antibody library.

By using different elution conditions, the method allows an approximatequantification of the binding affinity of the monoclonal antibody forits binding partner. Elution agents that may be used include chaotropicagents such as guanidine hydrochloride or urea at concentrations between10 μM and 8 M or ethylene glycol in an aqueous solution of 0.01% to 100%w/v. Elutions may also be carried out using aqueous or non-aqueoussolutions of glycine. Elutions may be carried out using aqueous or nonaqueous solutions of triethylamine between 1 μM and a saturatedsolution, preferably 100 mM, at a pH of between pH 8 and pH 13,preferably pH 11.5.

Storage and Shipping of Antibody Libraries and Harvested Antibodies

Ideally, the antibodies are pooled and stored until a time where it isdesired to isolate antigen specific antibodies. Antibodies can be storedat subzero degrees (e.g. liquid nitrogen, −70° C., or −20° C.). However,if the library is used for frequent isolation of antigen specificantibodies, it may be preferable to avoid freezing by storing thelibrary at temperatures of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C.Even though antibodies tend to be stable, repeated freeze/thaw circlesmay result in some degree of protein denaturation of the antibodies.

In an embodiment of the present invention the antibodies generated asdescribed herein above are stored in presence of one or more additivesselected from the group consisting of: antibiotics, anti-fungalcompounds, and buffer.

In another embodiment of the present invention the antibodies generatedas described herein above are stored at a temperature of about 0-100° C.

Antibodies according to the invention may furthermore be stored in thepresence of one or more additives such as: antibiotics, anti-fungalcompounds, antifreeze (e.g. glycerol), and buffer solutions. A preferredadditive is NaN₃—preferably 0.1% NaN₃ in PBS. Antibodies may even bestored at room temperature, preferably in the presence of preservatives,for relatively short period of times such as e.g. two weeks or less,preferably one week or less. This might be convenient e.g. in case oftransport of antibodies via shipping, mailing, etc.

In a preferred embodiment, antibodies are collected from the animalafter a period of at least 14 days, since this period of time is usuallyrequired to induce a detectable immune response in e.g. a chickensubsequent to immunization.

Ideally, the antibodies are pooled and stored until a time where it isdesired to isolate antigen specific antibodies. Antibodies can be storedat subzero degrees (e.g. liquid nitrogen, −70° C., or −20° C.). However,if the library is used for frequent isolation of antigen specificantibodies, it may be preferable to avoid freezing by storing thelibrary at temperatures in a range of about 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10° C. Even though antibodies tend to be stable, repeatedfreeze/thaw circles may result in some degree of protein denaturation ofthe antibodies.

The present invention furthermore relates to antibodies obtainable bythe methods herein.

The present invention furthermore relates to antibodies obtainable bythe methods herein.

Additional Embodiments

Another central aspect of the present invention relates to the use ofantibody libraries being specific to a multitude of antigens. Inparticular, the present invention thus relates to a method of isolatingantigen specific antibodies, wherein the method comprises the followingsteps:

-   -   immobilizing the antigen,    -   contacting the antibody library with the immobilized antigen        under conditions suitable for an antigen:antibody binding to        take place,    -   removing unbound antibody, and    -   recovering antigen specific antibodies.

An antibody library obtained after immunizing an animal with a peptidelibrary generated by random synthesis thus provides a unique possibilityof isolating antibodies with specificity to an antigen within a veryshort period of time—in theory within few hours.

Various methods of immobilizing the antigen can be employed inconnection with isolation of antigen specific antibodies. A preferredsolid phase is CNBr carrier. A preferred coupling is Epoxy coupling,since this approach is relatively cheap. Ultrasound sonification oracidic conditions may e.g. be used for breaking the antigen:antibodybinding in order to recover antigen specific antibodies. In general,various methods of coupling proteins to a carrier and subsequentlybreaking protein:protein interactions are well known in the art.

Finally, the present invention relates to antibodies obtainable by themethods herein.

The bond between antigens and antibodies is dependent on hydrogen bonds,hydrophobic bonds, electrostatic forces, and van der Waals forces. Theseare all bonds of a weak, non-covalent nature, yet some associationsbetween an antigen and an antibody can be quite strong. Accordingly, theaffinity constant for antibody-antigen binding can span a wide range,extending from below 10⁵ mol⁻¹ to more than 10¹² mol⁻¹. Affinityconstants are affected by temperature, pH and solvent. Apart from anaffinity of an antibody for a ligand, the overall stability of anantibody-ligand complex is also determined by the valency of the antigenand antibody and the structural arrangement of the interacting parts.

Affinity constants can be determined for monoclonal antibodies, but notfor polyclonal antibodies, as multiple bonds take place betweenpolyclonal antibodies and their antigens.

Monoclonal antibodies can be raised by fusion of B lymphocytes withimmortal cell cultures to produce hybridomas. Hybridomas will producemany copies of the exact same antibody—an essential feature in thedevelopment of antibodies for diagnostic applications. As monoclonalantibodies react with one epitope on the antigen, they are morevulnerable to the loss of epitope through chemical treatment of theantigen than are polyclonal antibodies. This can be offset by poolingtwo or more monoclonal antibodies to the same antigen.

The specificity of monoclonal antibodies makes them extremely efficientfor binding of antigen within a mixture of related molecules, such as inthe case of affinity purification. Also, as compared to polyclonalantibodies, the homogeneity of monoclonal antibodies is very high.Accordingly, if experimental conditions are kept constant, results frommonoclonal antibodies will be highly reproducible.

It is an object of the present invention to generate antibodies asdescribed herein above.

In an embodiment of the present invention antibodies are obtained asdescribed herein.

Use of Abs

In an embodiment of the present invention antibodies are generated andused as described herein.

It is furthermore an aspect of the present invention to use theantibodies generated or harvest or both by the methods described hereinfor the treatment of diseases causing diarrhea. However, antibodies canbe used to fight all diseases caused by bacteria, viruses, parasites(uncertain), chemicals (uncertain) or other agents which crosses thestomach-colon (e.g. cystic fibrosis). The bacteria are hindered inproliferation and the severity and course of the disease is decrease bythe antibodies. Due to the re-immunization system it will be possible totarget all intern diseases as for example cancer, AIDS, cold etc. Ifopen to the idea several diseases can theoretically be treated.

When it comes to pigs we show a special attention to the followingagents causing diarrhea:

-   -   1. Clostridia    -   2. Cocciciosis    -   3. Colibacillosis (E. Coli)    -   4. PED    -   5. PRRS    -   6. Rotavirus    -   7. TGE

Additionally we show attention to the most widespread agent causingdiarrhoea in human beings. To mention are: Camphylobachter jejuni,Salmonella, Shigella, E. Coli. However, still we do not have a completelist of the most lucrative diseases.

EXAMPLES Example 1: Synthesis of a Peptide Library

A library of peptides with a length of 6 amino acids and of 15 aminoacids was synthesized using standard methods. The 20 most commonlyoccurring (excl. selenocysteine and pyrrolysine) natural amino acidswere employed in each reaction and the library thus synthesized at leastin theory comprises 20⁶ or 20¹⁵, respectively, different peptides.

Example 2: Antigen Coupling

Method 1:

Peptides to be used as antigens are coupled to a carrier. In this methodthe peptide sequence must contain at least one cysteine residue or anNH2 group in order for the cross linking agent (MBS) to efficientlycouple the peptides to the carrier protein Keyhole Limpet Hemocyanin(KLH). In this example, a cysteine residue was added to the peptides asthe seventh residue before carrier conjugation.

Sephadex G-25 fine beads were swelled overnight in 5 ml buffer (0.1 MNaH2PO4 pH 6.0)/g of dry beads at room temperature. The swelled resinwas washed 4 times using 0.1 M NaH2PO4 pH 6.0 and poured into a 130×20mm column. The column was subsequently equilibrated with 15 ml of 0.1 MNaH2PO4 pH 6.0 and the resin was allowed to settle.

4 mg KLH was dissolved in 0.3 ml of 0.1 M NaH2PO4 pH 6.0. 1 mgm-Maleimidobenzoyl-N-Hydroxysuccinimide ester (MBS) in 150 μl ofDimethylformamide (DMF) was added drop-wise to the KLH solution understirring. The KLH/DMF mixture was incubated at room temperature for 30min on a rotating mixer.

1 ml of 0.1 M NaH2PO4 pH 6.0 was added to the KLH-MBS conjugate followedby thorough mixing. The mixture was subsequently loaded onto theSephadex G-25 column. 0.1 M NaH2PO4 pH 6.0 was used as column buffer.

1 ml fractions were collected by gravity flow in 1.5 ml tubes. Thefractions comprising the KLH-MBS conjugate (monitored at 280 nm) werepooled in a 15 ml conical tube.

200 μl of Dimethylformamide (DMF), 1 ml of peak fractions at 280 nm pH6.0, and 5 mg of the cysteine-containing peptide were mixed.

The peptide solution was mixed with the KLH-MBS conjugate and incubatedfor 12 to 16 hr at room temperature on a rotating mixing wheel.

The KLH-MBS-peptide conjugate was dialyzed for 24 hr at 4° C. against 1liter of PBS including two changes of buffer. Molecular weight cut offof the dialysis tube was 3,500.

Method 2:

By using Chicken Serum Albumin (CSA) or beads for coupling of theantigens an immune response against the carrier is avoided, therebyoptimizing the productivity of the chicken immune response. The couplingbetween the peptides and the carrier is mediated by glutaraldehyde,which couples free amino groups (—NH2) and cysteines. The ratio betweenpeptide and carrier has to be exact to avoid over-coupling. Bydecreasing the pH to a value lower than the pK value of the amino acidresidues, coupling to the side chains of the amino acid residues isavoided. Corrections in this calculation have to be made for sequencescontaining cysteine.

-   -   1. 5 mg/ml of peptide in PBS (Phosphate Buffered Saline).    -   2. CSA carrier protein is added in the ratio of 1-2 mol of        peptide per 50 amino acids in the carrier. Mix by magnetic        stirring under ventilated conditions.    -   3. While stirring add 0.2% glutaraldehyde in PBS, at a ratio of        1 ml glutaraldehyde/PBS per 20 μmol peptide/protein solution.    -   4. Incubate 1 hour at room temperature.    -   5. Add 1 M glycerine in PBS, pH 7.2, to a final concentration of        200 mM. Stir for 1 hour.    -   6. Separate the peptide-carrier from unbound peptide by dialysis        against PBS. 4 changes of buffer each 2 hours.

Example 3: Immunization

Method 1:

20 and 35 weeks old fowls of the “white Italian” race were immunized onday 0, 10, 20, 100 and 200. Blood samples and eggs were taken on day 0,7, 14, 21, 28, 35, 42, 49 and antibody concentration (IgY) was measuredusing ELISA techniques (se in the below).

The composition used for each immunisation was: 10 μg KLH-peptidedissolved in 0.01 M NaCl buffer (pH 7.2) to a volume of 500 μl and 500μl Specol to a total volume of 1 ml.

Method 2:

The chickens are immunized in the breast musculature with a pistol. Theanimals are immunized at day 1, 10 and subsequently every 4^(th) week.For each immunization one of the following compositions is used:

-   -   1. a minimum of 0.5 mg peptide-carrier mixed in the ratio of 1:1        to Freund's Incomplete Adjuvant diluted in PBS to 200    -   2. a minimum of 0.5 mg peptide-carrier mixed in the ratio of 1:1        to Super Adjuvant diluted in PBS to 200 μl (for the composition        of the Super Adjuvant, please see in the below).

Example 4: Testing of Super Adjuvant

Components of the Adjuvant:

-   -   1. Specol: marcol 52®, Span 85®, Tween 85® (tested with positive        results)    -   2. Vitamin E    -   3. Al(OH)₃    -   4. Heat Shock Proteins    -   5. CpG-peptides    -   6. Heat-inactivated B. Pertussis    -   7. dsRNA        Experimental Design:

7 groups of 4 chickens are immunized with the following Adjuvantcomponents:

1 2 3 4 5 6 7 Antigen Antigen Antigen Antigen Antigen Antigen AntigenSpecol Specol Specol Specol Specol Specol Specol E-vitamin E-vitaminE-vitamin E-vitamin E-vitamin E-vitamin Al(OH)3 Al(OH)3 Al(OH)3 Al(OH)3Al(OH)3 HSP HSP HSP HSP CpG CpG CpG B. Pertussis B. Pertussis Syntheticpl:CReagents:

-   -   1. PBS    -   2. Specol:        -   1. 8.1 ml Span 85® is mixed with 6.1 ml Tween 85®        -   2. 99 ml marcol 52® is mixed with 11 ml Span85®/Tween85®    -   3. Al(OH)₃: 8 mg/ml Al(OH)₃ solution is made (5-10 ml)        -   1. Couple peptide to Al(OH)₃: 440 μg peptide in 2.2 ml PBS            is added 2.2 ml (17.6 mg) 8 mg/ml Al (OH)₃.        -   2. Mix for 30 min. at 20° C.    -   4. HSP70 (Heat Shock Portein):        -   1. Coupling of peptide to HSP70:            -   Binding Buffer: PBS, 1 M ADP, 1 mM MgCl₂.            -   Stock: 704 μg HSP70 is mixed with 211 μg peptide in 1760                μl Binding Buffer            -   Incubate for 30 min at 37° C.    -   5. CpG-peptide:        -   1. ODN1826 is mixed to a concentration of 100 μg/ml (2×200            μg+2 ml water)    -   6. B. Pertussis:        -   1. mix 4×10¹⁰ in 2 ml PBS (2×10⁹/dosis)    -   7. dsRNA:        -   1. P(I:C) is mixed to a concentration of 100 μg/ml in 1 ml            PBS.            Adjuvant Mixture            Oil Mixture:    -   Group 1: 2.2 ml Specol    -   Group 2-7: Mix 21 ml Specol and 9 ml Vitamin E (Oil-in-Oil).        Make 12 aliquots of 2.2 ml each.        Aqueous Solution:    -   Group 3: Al(OH)₃:420 μl in 10 tubes (4.2 ml). Add 2×350 μl PBS        to tube 3a and 3b    -   Group 4: HSP70: Add 200 μl to 8 tubes (1600 μl). Add 300 μl PBS        to tube 4a and 4b.    -   Group 5: CpG: Add 400 μl to 6 tubes (2400 μl). Add 200 μl PBS to        tube 5a and 5b.    -   Group 6: B. Pertussis: Add 400 μl to 4 tubes (1600 μl). Add 100        μl PBS to tube 6a and 6b.    -   Group 7: dsRNA pl:C: Add 400 μl to 2 tubes (800 μl).        Mixture

The aqueous solutions (1.8 ml/tube) are added drop by drop duringvortexing to 2.2 ml oil mixture. Each tube contains immunizationmaterial for 4 chickens.

Immunization Protocol:

-   -   Day 1: 1. immunization of 4 chickens×7 groups with 1 ml        adjuvant.    -   Day 10: 2. immunization of 4 chickens×7 groups with 1 ml        adjuvant.

The mixtures are vortexed prior to use.

Blood samples are taken prior to each immunization and 14 days after thelast immunization.

According to the results of the above experiments the relevant adjuvantscomponents are chosen to be used in the final protocol to generate theantibody library.

Example 5: IgY ELISA Measurements

-   -   1. 1 μg/ml protein per well in 100 μl 10 mM phosphate buffer pH        7.4, containing 0.15 M NaCl (PBS), incubate for 2 h at room temp        and then at 4° C. for 16 h.    -   2. Wash 3 times with PBS containing 0.1% BSA (bovine serum        albumin) and 0.05% Tween 20.    -   3. Saturation with 200 μl PBS containing 2 mg/ml BSA for 3 h at        room temperature.    -   4. Standard curve 0.005 to 1.28 μg/ml, Serum fivefold diluted no        sample.    -   5. Sample diluted in 100 μl PBS containing 0.1% BSA and 0.05%        Tween is dispensed into wells and placed at 4° C. for 16 h.    -   6. Wash 3 times with PBS containing 0.1% BSA and 0.05% Tween 20.    -   7. Add secondary antibody at a ratio of 1/10000 to the wells and        incubate for 2 h at room temperature.    -   8. Wash with PBS containing 0.1% BSA and 0.05% Tween 20.    -   9. OPD Tablets.    -   10. Stop reaction by addition of sulfuric acid.    -   11. Measure absorbance at 450 nm.

Example 6: Antibody Purification

The peptide, protein, or chemical which the customer wants antibodiesagainst is synthesized and coupled to an epoxy bar. Following thecoupling the bar is transferred to the library. In the library relevantantibodies bind the antigens. By subsequent transfer to varying pHstrength the bound antibodies can be divided into three categories:weak, medium and strong binding. The antigen is bound covalently to thebar while the antibody is linked to the antigen by ion-bonds. Theweakest bound antibody is reused in the library.

Example 7: Antibody Isolation from the Egg Yolk

Classical Method with Ammonium Sulphate

-   -   1. Separate egg white and yolk. Wash the yolk with 2 volumes        H₂O.    -   2. Puncture the yolk and dissolve it in 1:10 weight/volume H₂O.    -   3. Freeze and dry the solution.    -   4. Add solid ammonium sulphate to 25% saturation; incubate at        room temperature for 20 min.    -   5. Centrifuge for 30 min at 2500×g.    -   6. Transfer the supernatant to a new glass and add solid        ammonium sulphate to 40% saturation. Incubate at room        temperature for 20 min.    -   7. Centrifuge for 30 min at 2500×g.    -   8. The pellet is redissolved in 1 ml PBS/0.1% NaN₃

Amount of ammonium sulphate (in grams) that has to be added a 1 Lsolution at 20° C.=553(S₂−S₁)/100−0.3*S₂, wherein S₂=Final concentrationof 25%; S₁=Start concentration, fx. 0%-25%: 133.25 g/L and 25%-40%:90g/L

Alternative Method 1 with NaCl

-   -   1. Separate egg white and yolk. Wash the yolk with 2 volumes        H₂O.    -   2. Puncture the yolk and dissolve it in 1:10 weight/volume H₂O.    -   3. Freeze and dry the solution.    -   4. Add 133.25 g solid NaCl to 25% saturation, incubate at room        temperature for 20 min.    -   5. Centrifuge for 30 min at 2500×g.    -   6. Transfer the supernatant to a new glass and add solid NaCl to        40% saturation. Incubate at room temperature for 20 min.    -   7. Centrifuge for 30 min at 2500×g.    -   8. The pellet is redissolved in 1 ml PBS/0.1% NaN₃        Alternative Method 2 with Chloroform    -   1. The egg yolk is separated from the egg white.    -   2. Add 100 mM Sodium Phosphate Buffer pH 7.6 to volume 60 ml and        mix.    -   3. Add 40 ml chloroform and mix to a semi-solid phase.    -   4. Centrifuge for 30 min. at 1500 g.    -   5. Isolate the supernatant.    -   6. PEG-6000 is added to a final concentration of 12% w/v. Mix        until PEG is dissolved.    -   7. Centrifuge for 10 min. at 16.000 g.    -   8. The pellet is dissolved in 100 mM Sodium Phosphate Buffer pH        7.6.    -   9. Sodium azide is added to 0.05%.

Example 8: Isolation of Antigen Specific Antibodies

One gram of cyanogen bromide-activated sepharose 4B (Pharmacia) wasswollen according to manufacturers instructions to yield 3.5 ml ofresin.

2.5 mg peptide was dissolved in 0.5 M NaCl, 0.1 M NaHCO₃, pH 8.3 andadded to the swollen sepharose. The peptide:resin mixture was mixed for2 hours at room temperature and centrifuged at 40×g for 5 min. The resinwas subsequently incubated with blocking buffer (0.2 M Glycine, pH 8.0)at 4° C. for 16 h. The peptide:resin was then poured into a column andwashed 5× with 0.5 M NaCl, 0.1 M NaHCO₃, pH 8.3 followed by 5 ml 0.5 MNaCl, 0.1 M ammonium acetate, pH 4.0.

After rinsing the column with 50 ml PBS, 8 ml IgY obtained in Example 3was mixed with 42 ml PBS and loaded overnight at 4° C. at a flow rate of30 μl/min. The column was subsequently washed with 50 ml PBS. Theantibodies that did not bind to the column were dialyzed for 24 hragainst 1 liter of PBS with 2 buffer changes 4° C. Cut off: 3,500. Afterdialyzation, antibodies can be returned to library to be used foranother occasion.

Antigen specific antibodies were eluted from the column with 0.15 MNaCl, 0.2 M glycine, pH 2.2. The eluate was neutralized with 1 MTris-HCL pH 8.0 and stored at 4° C. with 0.01% NaN₃.

Example 9: Affinity of Antigen Specific Antibodies

To measure the antibody affinity, purified antibody is reloaded oncolumn. Instead of eluting with pH 2.2 a range from pH 8.0 to pH 1.0 isused. Amount of antibody released at a specific pH is measured at 280nm. The absorbents peak of the pH range indicates its affinity.

Example 10: Optimal Conditions for Shipment and Storage of theAntibodies

Antibodies are very stable under mild denaturating conditions. Thismakes it possible to ship and store the antibodies in PBS Buffer, pH 7.0(the antibody structure is preserved) and sodium azide 0.05%(anti-bacterial and anti-fungal) at room temperature for at least 14days.

Storage at −20° C. is possible for years. The library will be dividedinto a stock at −20° C. and a stock at 1° C. to avoid damage due tothawing and freezing. Long-term storage of hybridoma cell lines for thegeneration of monoclonal antibodies is carried out by centrifugation at4° C. in a solution containing nourishment and DMSO (as cryoprotectant). Afterwards the hybridomas are slowly frozen and stored inliquid nitrogen.

Example 11: Monoclonal Antibodies

Removal of Bursa of Fabricius

The chickens will be killed by decapitation and the bursas ofFabricius/Bursa fabricii removed.

Bursa fabricii is removed using sterile techniques and transferred to acell culture dish, containing 10 ml 37° C. medium without serum.Non-bursa tissue is removed. Bursa fabricii is comminute/shredded with a19 G needle and subsequently with a pipette. The cell suspension istransferred to a sterile centrifuge bottle. The cells are allowed tosettle for app. 2 min. The supernatant is transferred to sterilecentrifuge bottles.

Fusions

Interspecies hybridomas are made between the antibody producing B-cellsof the chicken and a sp2/0 myeloma cell line as follows:

Add 0.3 g 50° C. polyethylene glycol (PEG) to 0.7 ml medium withoutserum and transfer to a 37° C. water bath. Centrifuge the B-cells at 400g for 5 min and remove the supernatant. Centrifuge 20 ml myeloma cellsat 400 g for 5 min and remove the supernatant. Resuspend the cellpellets in 5 ml medium without serum. The B-cells and the myeloma cellsare centrifuged at 400 g for 5 min. Remove the supernatant. Add 0.2 mlPEG to the respective cell pellets and resuspend the pellets carefully.Centrifuge at 400 g for 5 min. Resuspend the respective pellet in 5 mlmedium without serum and add 5 ml medium with 20% serum. Centrifuge at400 g for 5 min. Resuspend the cell pellets in medium containing 20%serum, 1×OPI Oxaloacetat Pyruvat Insulin, 1×AH Azaserin Hypoxanthin.

Isolation of Antibody Producing Cells

The antibodies are displayed on the surface of the hybridoma. Thereforethe hybridomas can be isolated as the antibodies are isolated aspreviously described. When the antibody producing hybridomas areisolated a fast production can be made.

The isolation must be carefully performed. Contamination from othercells cannot be tolerated in the production of monoclonal antibodies.The sonication has to be performed with a lower intensity, to avoiddestruction of living cells. When a single cell is isolated theprocedure as described in monoclonal antibody production is resumed. Theprobability of mistaken B-cell isolation is low, but will although betested in ELISA.

Alternatively the hybridomas can be isolated based on the secernation ofthe antibodies to the medium. Identification of the antibody producinghybridomas is made by analysis of the supernatant and further subcloningof the cells. After 5-6 subcloning the antibody producing monoclonalcell will be identified and isolated.

Monoclonal Antibody Production

Hybridomas are grown in Sigmas H4409 medium. The cells are divided in tofractions. The first fraction is stored frozen for later use. The secondfraction is grown to confluence and antibodies are collected when theantibody production ceases (9 days). It is expected that the antibodyproduction is 175 μg antibody/ml medium in 9 days.

By experience it is known that problems in the production of monoclonalantibodies primarily relate to randomised fusions. Some of thehybridomas grow fast, some slow, some do not grow at all. But if thefusions take place randomly, the probability of presentation of allkinds of peptides will increase by using more cells and more fusions. Ifa representative mix of hybridomas is achieved, it is possible toisolate one of each type of antibody producing hybridoma. The cells canbe isolated as a gathered group of cells producing the same antibody.Later in the growth-phase a natural selection will be made where thefast growing cells will dominate. At this stage it is tested if theantibody production is efficient.

Example 12: Diversity of Antibody Library

10.000 peptides of 15 amino acid residues in length were randomlysynthesized and used as the antigen library (Bib). Also 4 controlpeptides of 15 residues in length were synthesized: C, Ec, Sf-2, Sf-3.All peptides were C-terminally “tagged” with cysteine and coupled to thecarrier protein KLH. Afterwards the peptides were mixed to make a1:12.000 representation of all peptides: 2 mg Bib and 0.2 μg of eachcontrol peptide. The mixture was injected to 19 week old Isababcockhybrid chickens, a total of 2 mg peptide-KHL was used. Specol was usedas adjuvant at a ratio of 1:1. The chickens were reimmunized (boosted)on day 10 after the first immunization.

The control peptides are of the following sequences:

1. C: CREGPTKGMHTAVQGL (SEQ ID NO: 1) 2. Ec: MAPGSTVGTLVANMTC(SEQ ID NO: 2) 3. Sf-2: CDKIDSYAQQDLKKGLHLYG (SEQ ID NO: 3)4. Sf-3: CFFAYSDKIDSYAQQD (SEQ ID NO: 4)Test Results:

Prior to immunization blood samples were taken (pre-serum). Date: 22/3,4/4 and 18/4. The samples were diluted 1:100 and tested by ELISA. A1:10.000 dilution of secondary antibody-HRP was used. In these tests,each well was pre-coated with 100 ng peptide or peptide library.Controls were performed on wells not precoated with any protein orpeptide. For all measurements control measurements were performed.Measurements of blank samples are subtracted the sample measurements.All results have been reproduced. The highest obtained background signal(triple measurements) was subtracted from the ELISA results of thesamples. The background signals were registered 18/4. Before this thesample results was lower or equal to the highest background signal.

ELISA Results:

TABLE 1 Induction of immune response in a hen to selected targetantigens. The hen was immunized with a large amount and number ofdifferent peptides. Control samples were blood samples collected beforeimmunization. The results shown in the table are reproducible. Dato KLHBib Bib C C Ec Ec Sf-2 Sf-2 Sf-3 Sf-3 22/3 0 0 0 0 0 0 0 0 0 0 0  4/4 00 0 0 0 0 0 0 0 0 0 18/4 2.52 1.84 1.90 0.05 0.04 0.04 0.04 0.07 0.070.01 0.01Interpretation of the Results:

It is possible to immunize with 10.000 peptides and still obtainantibodies against a specific peptide. To conclude: it is possible todistribute the antibody production over as many as 10.000 antigens. Thehigh-quantity production of antibodies in a chicken can therefore beexploited to produce small quantities of a single antibody.

Contrary to the general presumptions in the art, it is possible toimmunise with large numbers of antigens, here 10,000 different peptides,AND obtaining specific antibodies. It is thus possible to produce alarge amount of different antibodies in one animal and by exploiting theability of the hen to produce large amounts of antibodies which can becollected from the eggs. It is furthermore demonstrated thatimmunisation with small amounts of antigen is possible without inducinganaphylactic shock in the animal.

Note:

The ELISA measurements can be translated into linear results by thestandard curve shown in FIG. 1. The curve is based on a number ofmeasurements on samples of known antibody concentration. The curve doesnot correspond to the original measurements and therefore it can only beused as an indication. The highest measurement (2.52 for KLH) liesoutside the standard curve. This can only be interpreted as high. Bib1.84 and 1.90 lie on the curve which correspond to an around 1000 ng/ml.

Example 13: Library Antibodies for the Treatment of Diarrhea inPiglets—Protocol I

Diarrhea in piglets is considered as one of the largest contributors(4-7% of all born piglets die of this) to the high mortality in piglets.The purpose of this experiment is to produce antibodies in chickensagainst pathogens causing diarrhea in piglets:

Clostridia

Cocciciosis

Colibacillosis (E. Coli)

Porcine Epidemic Diarrhoea (PED)

Porcine Reproductive and Respiratory Syndrome (PRRS)

Rotavirus

Transmissible Gastro-Enteritis (TGE)

Protocol I:

19 weeks old chickens, Gallus gallus domestica, are immunized withfragments from the pathogens (killing by heat and use all the juice) ina Super Adjuvant mixture.

100 chickens are immunized according to the scheme. Approximately 14chickens/group. The chickens are kept isolated to ensure propercollecting of eggs.

Experimental Period: 6 Weeks

Immunization Scheme:

Group 1 2 3 4 5 6 7 Pathogen Clostridia Cocciciosis Colibacillosis PEDPRRS Rotavirus TGE (E. Coli)Immunization: day 0Immunization: day 14Blood samples are drawn from the chickens at day 0, 10, 20, 30. Eggs arecollected from day 21.Evaluation of the immune response raised in chickens

The serum is isolated from the chickens, and antibody titers against thespecific pathogens determined by ELISA.

Purification of IgY

The specific antibodies are purified from the egg yolks usingNaCl-precipitation according to the example above. Hereafter a specificpurification of the antibodies is made by immune specific affinitychromatography.

Example 14: Library Antibodies for the Treatment of Diarrhea inPiglets—Protocol II

The antibody content in the sows' milk contributes to the protection ofthe piglet including both resistance to infection and development ofimmunological tolerance to harmless environmental antigens. The listedpathogens are common in the environment of the swine farm, and theantibodies can be isolated from milk from the sow. It is importantthough that the sows have not been protected from this environment as isoften the case. The main reason for the high mortality among piglets isthe lack of antibodies transferred from the mother through thecolostrum. Isolation of antibodies against top 10 diseases causingdiarrhea in piglets is performed using standard purification procedures.

Isolation of Peptide/Antigen

The peptides are magnetically coupled and the antibodies are dipped intothe peptide mixture. Subsequently the peptides are eluted by theirdegree of affinity as this correlates with decreasing pH during thedifferent elution steps.

The peptides thus captured are analyzed and synthesized in largequantities after which chickens are immunized with the newly synthesizedpeptides in larger. In this manner larger volumes of more specificantibodies may be generated and isolated. Furthermore, by analyzing thepeptides caught a subtraction method of selecting for mutated pathogenscan be employed.

Development of Specific Antibodies in Chicken

19 weeks old chickens, Gallus gallus domestica, are immunized withpeptides reacting with specific antibodies in a superadjuvant mixture.Hereby a specific immune response and specific antibodies are formed inthe chicken. The IgY antibodies against the pathogen/peptide arepurified from the egg yolk as described elsewhere.

Alternatively the antibodies can be isolated from the antibody libraryby coupling the identified peptides and/or the pathogen of interest.This method is already described.

Application of Antibodies

The pathogens described in this field exert their effect through thegastrointestinal tract. The application of antibodies may trap theantigen/pathogen in the gastrointestinal tract

-   -   thereby hinder the adherence of the pathogen and further        replication. Preferably this could decrease the duration and the        extent of the disease—thereby decrease the high mortality among        piglets.

The antibodies are administered to the piglets in two different ways:

-   -   Coupling of antibodies 1:1 by binding of the Fc portion of two        (or more) antibodies. The first antibody will trap the complex        after binding to the corresponding epitope, hereafter the second        antibody is able to bind another epitope creating a polyclonal        antibody-antigen complex inactivating the pathogen    -   Coupling of antibodies to a non-digestible material as clay or        plastic The antibodies trap/immobilize the pathogen and due to        the non-digestible material they will be transported through the        gastrointestinal tract and out with the feces.

REFERENCES

-   B. T Bennett et al., Review of polyclonal antibody production    procedures in mammals and poultry, ILAR News, 37, 93, 1995.-   J. Kollerup et al., Effect of homogenization and pasteurization on    the allergenicity of bovine milk analysed by a murine anaphylactic    shock model, Clin. Allergy, 17, 5, 449-458, 1987.-   J. Hau et al., Reagin production in mice: effect of subcutaneous and    oral sensitization with untreated bovine milk and homogenized bovine    milk, In Vivo, 3, 271, 1989.

What is claimed is:
 1. A method for generating a library of randomlygenerated IgY antibodies for targeting multiple ligands, said librarycomprising a plurality of antibodies, said method comprising the stepsof: a) generating a peptide library by random peptide synthesis, whereinthe resulting peptides are randomized at each amino acid residue; b)immunizing each of a plurality of avian organisms of the same ordifferent species with a composition comprising a plurality of differentpeptides of the library produced in step a); c) isolating antibodiesfrom the yolk of eggs laid by the plurality of immunized avian organismsof step b); and d) pooling said isolated antibodies.
 2. The method ofclaim 1 further comprising the steps of e) targeting said pool ofantibodies with a ligand; and f) isolating a library of antibodieshaving affinity for said ligand.
 3. The method of claim 1, wherein theligands are not used for immunization.
 4. The method of claim 1,wherein, in step b), each avian organism is immunized with at leastabout 100 different peptides.
 5. The method of claim 1, wherein, in stepb), a total of from 10 to 100000 avian organisms are immunized.
 6. Themethod of claim 5, wherein between 600 and 7000 avian organisms areimmunized.
 7. The method of claim 1, wherein the avian organism is abird of the gallinacean type.
 8. A method for isolating from the yolk ofeggs laid by a plurality of immunized avian organisms one or more IgYantibodies with affinity for a ligand without prior immunization of saidavian organism with said ligand, said method comprising the steps of: a)generating a library of IgY antibodies by the method of claim 1 to yielda pool of IgY antibodies; b) targeting said pool of antibodies with theligand; and c) isolating from the yolk of eggs laid by a plurality ofimmunized, avian organisms one or more IgY antibodies with affinity forthe ligand without prior immunization of said avian organisms with saidligand.
 9. The method of claim 8, wherein each avian organism isimmunized with at least about 100 different peptides.
 10. The method ofclaim 8, wherein a total of from 10 to 100000 avian organisms areimmunized.
 11. The method of claim 10, wherein a total of between 600and 7000 avian organisms are immunized.
 12. The method of claim 8,wherein the avian organism is a bird of the gallinacean type.